Yongming Qiu

Selective ablation of tumor-associated macrophages suppresses metastasis and angiogenesis

Tumor‐associated macrophages (TAM) play a critical role in promoting tumor development and metastasis. In the present study, we found that legumain, an asparaginyl endopeptidase, was highly expressed on the surface of TAM. A doxorubicin‐based prodrug specifically activated by legumain selectively ablated TAM and resulted in a significant reduction of angiogenic factors and related tumor vessel growth. Treatment with the prodrug also suppressed circulating tumor cells and myeloid immune suppressor Gr‐1+/CD11b+ cells in tumor‐bearing animals. After selective ablation of TAM using the prodrug, tumor growth and metastases were greatly inhibited in murine tumor models. These results indicate that legumain‐activated prodrugs targeting TAM in tumors might represent a novel anticancer strategy.

Functional Role of Asparaginyl Endopeptidase Ubiquitination by TRAF6 in Tumor Invasion and Metastasis

BACKGROUND Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive. METHODS We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays. The sera of healthy donors and breast cancer patients were examined by enzyme-linked immunosorbent assay, and a tissue array of 314 breast cancer specimens was assessed for AEP and TRAF6 by immunohistochemistry. Furthermore, the effects of AEP inhibitors or monoclonal antibodies on pulmonary metastasis were evaluated in murine models. The statistical significance between groups was determined using two-tailed Student t tests. RESULTS We demonstrate that TRAF6 ubiquitinates the proform of AEP through K63-linked polyubiquitin, reversible by USP17, and forms a complex with HSP90α to subsequently promote pro-AEP intracellular stability as well as secretion. Disrupting the interaction between pro-AEP and TRAF6 or inhibiting HSP90α reduced pro-AEP secretion and consequently reduced tumor metastasis. Higher circulating AEP levels were detected in the sera of breast cancer patients, and AEP inhibitors or neutralizing antibodies remarkably decreased tumor metastasis in murine models. Notably, TRAF6 and AEP were overexpressed in human breast neoplasms and correlated with poor prognosis. Patients with low AEP/TRAF6 expression survived for a mean of 111 months (95% confidence interval [CI] = 108 to 115 months), whereas those with high AEP/TRAF6 expression survived for a mean of only 61 months (95% CI = 42 to 79 months; P < .001). CONCLUSIONS Our study elucidates a novel mechanism of AEP regulation and an alternative oncogenic pathway for TRAF6 in breast cancer, which suggests that AEP and TRAF6 protein levels may have prognostic implications in breast cancer patients. Thus, AEP may serve as a biomarker as well as new therapeutic target.

The Role of Extracellular Vesicles: An Epigenetic View of the Cancer Microenvironment

Exosomes, microvesicles, and other extracellular vesicles are released by many cell types, including cancer cells and cancer-related immune cells. Extracellular vesicles can directly or indirectly facilitate the transfer of bioinformation to recipient cells or to the extracellular environment. In cancer, exosomes have been implicated in tumor initiation, proliferation, and metastasis. Extracellular vesicles can transmit proteins and nucleic acids that participate in DNA methylation, histone modification, and posttranscriptional regulation of RNA. Factors transmitted by extracellular vesicles reflect the donor cell status, and extracellular vesicles derived from tumor cells may be also responsible for altering expression of tumor promoting and tumor suppressing genes in recipient cells. Thus, circulating extracellular vesicles may act as biomarkers of cancer, and detection of these biomarkers may be applied to diagnosis or assessment of prognosis in patients with cancer.

Overexpression of isocitrate dehydrogenase-1R132H enhances the proliferation of A172 glioma cells via aerobic glycolysis

Gliomas are the most common type of primary malignancy of the central nervous system. The identification of mutations in the gene encoding isocitrate dehydrogenase‑1 (IDH1) represents a key area of investigation in studies on glioma. The IDH1R132H mutation is a heterozygous point mutation, which affects the amino acid arginine at position 132, however, the metabolic importance of this mutation in tumor cell growth remains to be elucidated. In the present study, A172 glioma cell lines stably overexpressing either wild‑type IDH1 or IDH1R132H were produced. The results demonstrated that the IDH1R132H mutation enhanced the proliferation of the A172 glioma cells in vitro. Furthermore, IDH1R132H performed this function by elevating the expression levels of hypoxia inducible factor‑1α, leading to an increase in the expression levels of the key glycolytic enzymes, glucose transporter 1 and hexokinase 2. Therefore, the metabolism was shifted towards aerobic glycolysis, leading to an increase in glucose uptake and lactate production. These findings demonstrated that the IDH1R132H molecular target was involved in orchestrating the Warburg effect in mutant IDH1R132H glioma cells.

Overexpression of FoxO3a is associated with glioblastoma progression and predicts poor patient prognosis

Forkhead transcription factor FoxO3a has been reported to have ambiguous functions and distinct mechanisms in various solid tumors, including glioblastoma (GBM). Although a preliminary analysis of a small sample of patients indicated that FoxO3a aberrations in glioma might be related to aggressive clinical behavior, the clinical significance of FoxO3a in glioblastoma remains unclear. We investigated the expression of FoxO3a in a cohort of 91 glioblastoma specimens and analyzed the correlations of protein expression with patient prognosis. Furthermore, the functional impact of FoxO3a on GBM progression and the underlying mechanisms of FoxO3a regulation were explored in a series of in vitro and in vivo assays. FoxO3a expression was elevated in glioblastoma tissues, and high nuclear FoxO3a expression in human GBM tissues was associated with poor prognosis. Moreover, knockdown of FoxO3a significantly reduced the colony formation and invasion ability of GBM cells, whereas overexpression of FoxO3a promoted the colony formation and invasion ability. The results of in vivo GBM models further confirmed that FoxO3a knockdown inhibited GBM progression. More, the pro‐oncogenic effects of FoxO3a in GBM were mediated by the activation of c‐Myc, microtubule‐associated protein 1 light chain 3 beta (LC3B) and Beclin1 in a mixed‐lineage leukemia 2 (MLL2)‐dependent manner. These findings suggest that high FoxO3a expression is associated with glioblastoma progression and that FoxO3a independently indicates poor prognosis in patients. FoxO3a might be a novel prognostic biomarker or a potential therapeutic target in glioblastoma.

Bundling potent natural toxin cantharidin within platinum (IV) prodrugs for liposome drug delivery and effective malignant neuroblastoma treatment

Neuroblastoma (NB) is one of the most commonly seen malignancies in childhood and infancy. Cantharidin is a highly potent natural toxin that possesses potent anti-tumor properties on various cancers including NB. However, exposure to cantharidin can cause severe chemical burns and application of cantharidin for cancer therapy is limited. Here we report a strategy of bundling cantharidin within a hybrid platinum (IV) prodrug conjugate. This hydrophobic drug conjugate, ie, CanPt can be further formulated into liposome for drug delivery to minimize the exposure of cantharidin to normal cells for efficient chemotherapeutic agent against NB.

Suppression of asparaginyl endopeptidase inhibits polyomavirus middle T antigen-induced tumor formation and metastasis.

Elevated circulating asparaginyl endopeptidase (AEP), a novel lysosomal protease, has been found in breast cancer, and AEP is thus considered to be a prognostic factor in this disease. However, the pathological functions of circulating AEP in the development of breast cancer and the potential of AEP-targeted therapy remain unclear. We used MMTV-PyVmT transgenic mice, which spontaneously develop mammary tumors. Western blotting showed overexpression of AEP in both primary tumor tissue and lung metastases compared to their normal counterparts. Moreover, the concentration of circulating AEP gradually increased in the serum during the development of mammary tumors. Purified AEP protein injected through the tail vein promoted tumor growth and mammary tumor metastasis and shortened survival, whereas AEP-specific small compound inhibitors (AEPIs) effectively suppressed tumor progression and prolonged host survival. Further analysis of the molecular mechanism revealed that AEP was important for PI3K/AKT pathway activation. Thus, an elevated serum AEP level was closely related to mammary cancer progression and metastasis, and AEP is a potential target for breast cancer therapy in the clinic.

Expression and functions of glutamate and γ‑aminobutyric acid transporters in ischemic models

Glutamate and γ-aminobutyric acid (GABA) transporters serve central roles in normal neuronal activity and are associated with numerous pathological brain conditions, including ischemia and epilepsy. However, the interplay between these transporters in ischemia remains unclear. In the present study, the expression levels of the excitatory amino acid carrier 1 (EAAC1) and GABA transporter 1 (GAT1) were analyzed in vivo and in vitro within ischemic models by immunofluorescence, western blot and RT-qPCR. Cell survival rates were analyzed following altered expression of these transporters within neuronal cells by flow cytometry. Expression levels of EAAC1 were reduced within the cerebrum of focal cerebral ischemic middle cerebral artery occlusion rat models as well as in primary neurons cultured under hypoxia. However, GAT1 expression levels were slightly elevated under ischemic conditions. The altered expression levels of EAAC1 and GAT1 were combined within neuron cells and the effects were investigated. Apoptotic analysis revealed that EAAC1 suppression and overexpression of GAT1 increased neuronal cell apoptosis under hypoxic conditions; however, EAAC1 overexpression combined with GAT1 knockdown reduced neuronal cell apoptosis under hypoxic conditions. The present study detected the expression levels of the glutamate and GABA transporters under hypoxia, in association with ischemia. The results indicated that, increased expression of EAAC1 combined with GAT1 suppression may provide protective effects in the treatment of epilepsy and ischemia.

Tumor-Targeted Accumulation of Ligand-Installed Polymeric Micelles Influenced by Surface PEGylation Crowdedness

With respect to the intriguing biocompatibility and the stealthy functions of poly(ethylene glycol) (PEG), PEGylated nanoparticulates have been intensively engineered for utilities as drug delivery vehicles. To advocate the targeted drug transportation, targeting ligands were strategically installed onto the surface of PEGylated nanoparticulates. The previous in vitro investigations revealed that the ligand-specified cell endocytosis of nanoparticulates was pronounced for the nanoparticulates with adequately high PEG crowdedness. The present study aims to explore insight into the impact of PEGylation degree on in vivo tumor-targeted accumulation activities of cRGD-installed nanoparticulates. The subsequent investigations verified the importance of the PEGylation crowdedness in pursuit of prolonged retention in the blood circulation post intravenous administration. Unprecedentedly, the PEGylation crowdedness was also identified as a crucial important parameter to pursue the tumor-targeted accumulation. A plausible reason is the elevated PEGylation crowdedness eliciting the restricted involvement in nonspecific protein adsorption of nanoparticulates in the biological milieu and consequently pronouncing the ligand-receptor-mediated binding for the nanoparticulates. Noteworthy was the distinctive performance of the class of the proposed systems once utilized for transportation of the mRNA payload to the tumors. The protein expression in the targeted tumors appeared to follow a clear PEGylation crowdedness dependence manner, where merely 2-fold PEGylation crowdedness led to remarkably 10-fold augmentation in protein expression in tumors. Hence, the results provided important information and implications for design of active-targeting PEGylated nanomaterials to fulfill the targeting strategies in systemic applications.