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A novel pan-cancer biomarker plasma heat shock protein 90alpha and its diagnosis determinants in clinic.

Liu, W.; Li, J.; Zhang, P. (...)

A sensitive and specific diagnosis biomarker, in principle scalable to most cancer types, is needed to reduce the prevalent cancer mortality. Meanwhile, the investigation of diagnosis determinants of a biomarker will facilitate the interpretation of its screening results in clinic. Here we design a large-scale (1,558 enrollments), multicenter (multiple hospitals), and cross-validation (two datasets) clinic study to validate plasma Hsp90alpha quantified by ELISA as a pan-cancer biomarker. ROC curve shows the optimum diagnostic cutoff is 69.19 ng/mL in discriminating various cancer patients from all controls (AUC 0.895, sensitivity 81.33% and specificity 81.65% in test cohort; AUC 0.893, sensitivity 81.72% and specificity 81.03% in validation cohort). Similar results are noted in detecting early-stage cancer patients. Plasma Hsp90alpha maintains also broad-spectrum for cancer subtypes, especially with 91.78% sensitivity and 91.96% specificity in patients with AFP-limited liver cancer. In addition, we demonstrate levels of plasma Hsp90alpha are determined by ADAM10 expression, which will affect Hsp90alpha content in exosomes. Furthermore, Western blotting and PRM-based quantitative proteomics identify that partial false ELISA-negative patients secret high levels of plasma Hsp90alpha. Mechanism analysis reveal that TGFbeta-PKCgamma gene signature defines a distinct pool of hyperphosphorylated Hsp90alpha at Theronine residue. In clinic, a mechanistically relevant population of false ELISA-negative patients express also higher levels of PKCgamma. In sum, plasma Hsp90alpha is a novel pan-cancer diagnosis biomarker, and cancer diagnosis with plasma Hsp90alpha is particularly effective in those patients with high expression of ADAM10, but may be insufficient to detect the patients with low ADAM10 and those with hyperphosphorylated Hsp90alpha. This article is protected by copyright. All rights reserved.

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Biophysical and Biological Characterization of PEGylated Recombinant Human Endostatin (M2 ES).

Guo, L.; Xu, B.; Zhou, D. (...)

Recombinant human endostatin (MES), showing potent inhibition on angiogenesis and tumor growth, has great potential as therapeutic agents for tumor. The aim of this study was to evaluate the biophysical and biological characterization of PEGylated recombinant human endostatin (M2 ES). MES was mono-PEGylated by conjugation with methoxy polyethylene glycol aldehyde (mPEG-ALD), and the modification site was identified by digested peptide mapping and MALDI-TOF-MS. The purity was assessed by SDS-PAGE, HPLC, and capillary zone electrophoresis. The physicochemical property was analyzed through fluorescence spectroscopy, and circular dichroism. The bioactivity and anti-tumor efficacy of M2 ES were evaluated using an in vitro endothelial cell migration model and a nunll-mouse xenograft model of a prostatic cancer, respectively. M2 ES molecules contain a single 20 kDa mPEG-ALD molecule conjugated at the N-terminal portion of MES. The purity of M2 ES was greater than 98%. The physicochemical analysis demonstrated that PEGylation does not change the secondary and tertiary structure of MES. Notably, M2 ES retards endothelial cell migration and tumor growth when compared to control group. These biophysical and biological characterization study data contribute to the initiation of the ongoing clinical study. This article is protected by copyright. All rights reserved.

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eHsp90alpha and clusterin synergistically promote breast cancer epithelial-to-mesenchymal transition and metastasis via LRP1.

Tian, Y.; Wang, C.; Chen, S. (...)

Extracellular heat shock protein 90 alpha (eHsp90alpha) has been widely reported to promote tumor cell motility and tumor metastasis in various types of cancer. Several extracellular proteins and membrane receptors have been identified as interacting proteins of eHsp90alpha and mediate its pro-metastasis function. However, the regulatory mechanism of eHsp90alpha activity remains largely unknown. Here, we reported that clusterin, a novel interacting protein of eHsp90alpha, modulated eHsp90alpha signaling. We found that clusterin potentiated the effects of eHsp90alpha on the activation of AKT, ERK, NF-kappaB, epithelial-to-mesenchymal transition (EMT) and migration in breast cancer cells. Furthermore, in vivo investigations demonstrated similar synergistic effects of eHsp90alpha and clusterin on tumor metastasis. Notably, the effects of eHsp90alpha and clusterin were mediated by low-density lipoprotein receptor-related protein 1 (LRP1). Proximity ligation assay and co-immunoprecipitation experiments demonstrated that clusterin participated in the complex formation of eHsp90alpha and LRP1, which enhanced the binding affinity of eHsp90alpha to LRP1. Collectively, our data establish a role of clusterin as a novel modulator of eHsp90alpha, and unravel detailed molecular mechanisms underlying the synergistic metastasis-promoting effects of clusterin and eHsp90alpha.

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Quality, bioactivity study, and preclinical acute toxicity, safety pharmacology evaluation of PEGylated recombinant human endostatin (M2ES)

Guo, L.; Geng, X.; Liu, L. (...)

Endostar, a potent endogenous antiangiogenic factor, is wildly used in clinics. However, it was easily degraded by enzymes and rapidly cleared by the kidneys. To overcome these shortcomings, PEGylated recombinant human endostatin was developed. In this study, the purity of M2ES was evaluated by silver stain and reversed-phase high-performance liquid chromatography. Ultraviolet spectrum was used to examine the structural of M2ES and endostar. The bioactivity and antitumor efficacy of M2ES were evaluated using an in vitro endothelial cell migration model and athymic nude mouse xenograft model of a heterogeneous lung adenocarcinoma, respectively. A preclinical study was performed to evaluate the acute toxicity and safety pharmacology in rhesus monkeys. The purity of M2ES was more than 98%; PEG modification has no effect on endostatin structure. Compared with the control group, M2ES dramatically retards endothelial cell migration and tumor growth. After intravenous (IV) infusions of M2ES at a dose level of three and 75 mg/kg in rhesus monkeys, there was no observable serious adverse event in both acute toxicity and safety pharmacology study. On the basis of the quality and bioactivity study data of M2ES and the absence of serious side effect in rhesus monkeys, M2ES was authorized to initiate a phase I clinical trial.

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High expression of CCR5 in melanoma enhances epithelial-mesenchymal transition and metastasis via TGF beta 1

Liu, J.; Wang, C.; Ma, X. (...)

Chemokine receptors are highly expressed in various cancers and play crucial roles in tumor progression. However, their expression patterns and functions in melanoma are unclear. The present study aimed to identify the chemokine receptors that play critical roles in melanoma progression and unravel the underlying molecular mechanisms. We found that CCR5 was more abundant in melanoma cells than normal cells and was positively associated with tumor malignancy in clinical patients. Animal experiments suggested that CCR5 deficiency in B16/F10 or A375 cells suppressed primary tumor growth and lung metastasis, whereas CCR5 overexpression in B16/F0 cells enhanced primary tumor growth and lung metastasis. CCR5 played a critical role in proliferation and migration of melanoma cells in vitro. Importantly, CCR5 was required for maintenance of the mesenchymal phenotype of metastatic melanoma cells. Mechanistically, CCR5 positively regulated expression of TGF beta 1, which in turn induced epithelial-mesenchymal transition and migration via PI3K/AKT/GSK3 beta signaling. Collectively, our results establish a critical role of CCR5 expressed by melanoma cells in cancer progression and reveal the novel mechanisms controlling this process, which suggests the prognostic value of CCR5 in melanoma patients and provides novel insights into CCR5-targeted strategies for melanoma treatment. Copyright (c) 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Lysyl Oxidase-Like Protein 2 Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer

Wang, C.; Xu, S.; Tian, Y. (...)

Tumor lymphangiogenesis has been previously documented to predict regional lymph node metastasis and promote the spread to distant organs. However, the underlying mechanism initiating tumor lymphangiogenesis remains unclear. Here we described a novel role of tumor cell-derived Lysyl Oxidase-like protein 2 (LOXL2) in promoting lymphangiogenesis and lymph node metastasis in breast cancer. Immunohistochemistry (INC) analysis of samples from breast cancer patients showed that the expression of LOXL2 was positively correlated with lymphatic vessel density and breast cancer malignancy. In animal studies, LOXL2-overexpressing breast cancer cells significantly increased lymphangiogenesis and lymph node metastasis, whereas knockdown of LOXL2 suppressed both processes. In order to study the mechanisms of lymphangiogenesis progression, we performed further in vitro investigations and the data revealed that LOXL2 significantly enhanced lymphatic endothelial cells (LECs) invasion and tube formation through directly activation of the Akt-Snail and Erk pathways. Moreover, LOXL2 also stimulated fibroblasts to secrete high level of pro- lymphangiogenic factors VEGF-C and SDF-1 alpha. Taken together, our study elucidates a novel function of tumor cell secreted LOXL2 in lymphangiogenesis and lymph node metastasis, demonstrating that LOXL2 serves as a promising target for anti-lymphangiogenesis and anti-metastasis therapies for breast cancer.

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