To explore the clinical significance of seven diabetes-related serum microRNAs (miR-9, miR-29a, miR-30d, miR34a, miR-124a, miR146a and miR375) during the pathogenesis of type 2 diabetes (T2D), 56 subjects were recruited to this study: 18 cases of newly diagnosed T2D (n-T2D) patients, 19 cases of pre-diabetes individuals (impaired glucose tolerance [IGT] and/or impaired fasting glucose [IFG]) and 19 cases of T2D-susceptible individuals with normal glucose tolerance (s-NGT). Serum miRNAs were determined by real-time RT-PCR. Expression levels of single miRNAs and the expression signatures of miRNAs as a panel were analysed among the three groups. In n-T2D, all 7 miRNAs were significantly up-regulated compared with s-NGT and five were significantly up-regulated compared with pre-diabetes, while miRNA expression was not significantly different between s-NGT and pre-diabetes. By Canonical discriminant analysis, 70.6% of n-T2D subjects (12/17) were recognized by canonical discriminant function, while s-NGT and pre-diabetes subjects could not be discriminated from each other. Similar results were found in Hierarchical Clustering analysis based on the expression levels of all seven miRNAs. In different statistical analysis, miR-34a always showed the most significant differences. We conclude that the expression levels of seven diabetes-related miRNAs in serum were significantly elevated in n-T2D compared with pre-diabetes and/or s-NGT, and the latter two groups featured similar expression patterns of these miRNAs, suggesting that during the pathogenesis of T2D, the peripheral diabetes-related miRNAs have not changed significantly from s-NGT at pre-diabetic stage.

Tissue factor (TF) expression by tumor cells correlates with metastasis clinically and supports metastasis in experimental settings. However, the precise pathways coupling TF to malignancy remain incompletely defined. Here, we show that clot formation by TF indirectly enhances tumor cell survival after arrest in the lung, during experimental lung metastasis, by recruiting macrophages characterized by CD11b, CD68, F4/80, and CX 3 CR1 (but not CD11c) expression. Genetic or pharmacologic inhibition of coagulation, by either induction of TF pathway inhibitor expression or by treatment with hirudin, respectively, abrogated macrophage recruitment and tumor cell survival. Furthermore, impairment of macrophage function, in either Mac1-deficient mice or in CD11b-diphtheria toxin receptor mice in which CD11b-positive cells were ablated, decreased tumor cell survival without altering clot formation, demonstrating that the recruitment of functional macrophages was essential for tumor cell survival. This effect was independent of NK cells. Moreover, a similar population of macrophages was also recruited to the lung during the formation of a premetastatic niche. Anticoagulation inhibited their accumulation and prevented the enhanced metastasis associated with the formation of the niche. Our study, for the first time, links TF induced coagulation to macrophage recruitment in the metastatic process. (Blood. 2012;119(13): 3164-3175) IntroductionClinical and experimental studies over the past 30 years have established that the coagulation system actively supports tumor progression and metastasis. Consistent with these observations, expression of procoagulants by tumor cells, among them tissue factor (TF), cancer procoagulant, 1 and selectin ligands, correlates with advanced disease and poor outcome for multiple cancer types. 2,3 TF (also known as coagulation factor III or CD142) is the protease receptor that initiates coagulation after injury through the extrinsic pathway. Under normal physiologic conditions, TF expression is limited to extravascular sites that only become exposed to blood after trauma. In this case, the exposed TF binds to and activates the blood-borne coagulation factor FVII, triggering clot formation through a cascade of proteolytic events that results in thrombin formation, activation of platelets, and fibrin deposition. 4 In addition to triggering coagulation, the binding of FVIIa to TF activates intracellular signaling pathways through the TF cytoplasmic domain, by activating G-protein-coupled protease activated receptors (PARs), especially PAR2. 4 These signaling pathways support tumor angiogenesis 5,6 and regulate tumor progression. 7 Intracellular signaling pathways can be distinguished experimentally from the extracellular coagulative roles of TF by specific antibodies 7 or deletion of the cytoplasmic domain that eliminates many forms of TF signaling but still triggers coagulation. 6,8,9 TF enhances tumor growth and angiogenesis, [4][5][6][7]10 and specifically plays an important role in some experiment...

Liver metastasis from colorectal cancer is a leading cause of cancer mortality. Myeloid cells play pivotal roles in the metastatic process, but their prometastatic functions in liver metastasis remain incompletely understood. To investigate their role, we simulated liver metastasis in C57BL/6 mice through intrasplenic inoculation of MC38 colon carcinoma cells. Among the heterogeneous myeloid infiltrate, we identified a distinct population of CD11b/Gr1 mid cells different from other myeloid populations previously associated with liver metastasis. These cells increased in number dramatically during establishment of liver metastases and were recruited from bone marrow by tumor-derived CCL2. Liver metastasis of Lewis lung carcinoma cells followed this pattern but this mechanism is not universal as liver colonization by B16F1 melanoma cells did not recruit similar subsets. Inhibition of CCL2 signaling and absence of its cognate receptor CCR2 reduced CD11b/Gr1 mid recruitment and decreased tumor burden. Depletion of the CD11b/Gr1 mid subset in a transgenic CD11b-diphtheria toxin receptor mouse model markedly reduced tumor cell proliferation. There was no evidence for involvement of an adaptive immune response in the prometastatic effects of CD11b/Gr1 mid cells. Additionally, an analogous myeloid subset was found in liver metastases of some colorectal cancer patients. Conclusion: Collectively, our findings highlight the importance of myeloid cells-in this case a selective CD11b/Gr1 mid subsetin sustaining development of colorectal cancer liver metastasis and identify a potential target for antimetastatic therapy. (HEPATOLOGY 2013;57:829-839) M etastatic colorectal cancer (CRC) is a prominent cause of cancer mortality worldwide. 1 Hepatic metastases are found in approximately 15% of CRC patients at primary diagnosis 2 with 14% subsequently developing metastases.3 Development of new treatment modalities for CRC liver metastasis is urgently required and a greater understanding of the biology of this process will help establish new therapeutics aimed at downstaging the disease, improving operability, and prolonging survival.Metastasis is a multistep process involving complex and continuous interactions between tumor cells and the host microenvironment.4 Several myeloid-derived cell types have been shown to play key roles in the metastatic cascade, including intravasation, extravasation, 5 and colonization at secondary sites by stimulating tumor cell proliferation and angiogenesis and suppressing antitumor immunity.6-8 However, delineation of their roles in metastasis is complicated by the heterogeneity of myeloid phenotypes that appears to be both tumor-and organ-selective. Vascular endothelial growth factor receptor 1 (VEGFR1) þ hematopoietic progenitor cells accumulated at premetastatic sites to promote adherence and growth of lung Lewis carcinoma (LLC) and B16F1 tumor cells, 9 while a Mac-1 þ myeloid population with different markers was

Background & Aims Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis mainly due to metastasis. MicroRNAs are endogenous small noncoding RNAs that regulate cellular gene expression and are functionally linked to tumourigenesis. Using microarray analysis, we recently identified 20 miRNAs associated with HCC metastasis. Here, we carried out further analyses on one of these microRNAs, let-7g, to determine whether it is functionally linked to HCC metastasis. Methods Quantitative real-time polymerase chain reaction was used to determine the level of mature let-7g transcript in HCC clinical specimens and its correlation with patient survival. Ectopic expression of let-7g was carried out in HCC cell lines to assess its influence on cell growth, migration and invasion. Results We confirmed that the level of let-7g was significantly lower in metastatic HCCs compared to metastasis-free HCCs. Moreover, low let-7g expression in a tumour was predictive of poor survival in HCC patients. Functional studies indicated that ectopic expression of let-7g significantly inhibits HCC cell migration and cell growth. In-silico analysis revealed members of soluble collagens as potential targets of let-7g. Consistently, the levels of type I collagen α2 (COL1A2) and let-7g were inversely correlated in HCC clinical specimens. COL1A2 was experimentally validated as a direct target of let-7g. Moreover, addition of COL1A2 counteracted the inhibitory effect of let-7g on cell migration. Conclusions These results suggest that let-7g may suppress HCC metastasis partially through targeting COL1A2.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide and therapeutic agents for this malignancy are lacking. MicroRNAs play critical roles in carcinogenesis and present tremendous therapeutic potential. Here we report that microRNA-206 is a robust tumor suppressor that plays important roles in the development of HCC by regulating cell cycle progression and cMet signaling pathway. MicroRNA-206 was under-expressed in livers of two HCC mouse models, human individuals bearing HCC, and human HCC cell lines. Combining bioinformatic prediction and molecular and cellular approaches, we identified cMET (Met proto-oncogene), CCND1, and CDK6 as functional targets of microRNA-206. By inhibiting expression of cMET, CCND1 and CDK6, microRNA-206 delayed cell cycle progression, induced apoptosis and impaired proliferation of three distinct human HCC cell lines. Systemic administration of microRNA-206 completely prevented HCC development in both cMyc and AKT/Ras HCC mice, while 100% of control mice died from lethal tumor burdens. Conversely, re-introduction of cMet or Cdk6 into livers of cMyc and AKT/Ras HCC mice recovered growth of HCC inhibited by microRNA-206. These results strongly suggested that cMet and Cdk6 were two functional targets that mediated the inhibitory effect of microRNA-206 on the development of HCC. MicroRNA-206 overexpression demonstrated a profound therapeutic effect on HCC in xenograft and cMyc HCC mice. In summary, this study defines a potentially critical role of microRNA-206 in preventing the growth of HCC, and suggests its use as a potential therapeutic strategy for this malignancy.

Treatments of glioblastoma (GBM) have not been very effective, largely due to the inefficiency of drugs in penetrating the blood brain barrier (BBB). In this study, we investigated the potential of exosome-coated doxorubicin (DOX)-loaded nanoparticles (ENP DOX) in BBB penetration, inducing immunogenic cell death (ICD) and promoting survival of GBM-bearing mice. DOX-loaded nanoparticles (NP DOX) were coated with exosomes prepared from mouse brain endothelial bEnd.3 cells. ENP DOX cellular uptake was examined. Penetration of ENP DOX through the BBB was tested in an in vitro transwell system and a GBM mouse model. The effects of ENP DOX in inducing apoptosis and ICD were assessed. Finally, the efficacy of ENP DOX in the treatment of GBM-bearing mice was assessed. ENP DOX was taken up by bEnd.3 cells and could penetrate the BBB both in vitro and in vivo. In vitro, END DOX induced apoptosis and ICD of glioma GL261 cells. Systemic administration of ENP DOX resulted in maturation of dendritic cells, activation of cytotoxic cells, altered production of cytokines, suppressed proliferation and increased apoptosis of GBM cells in vivo and prolonged survival of GBM-bearing mice. Our findings indicate that ENP DOX may be a potent therapeutic strategy for GBM which warrants further investigation in clinical application. K E Y W O R D S blood brain barrier, doxorubicin, exosome-coated doxorubicin-loaded nanoparticle, glioblastoma, immunogenic cell death 1 | INTRODUCTION Glioblastoma (GBM) is a highly aggressive brain tumor with an extremely poor prognosis and a small rate (4%-5%) or 5-year survival. 1 Current treatments of GBM include surgeries, radiotherapy, and/or chemotherapy. These treatments not only cause severe side effects, but also only slightly improve the overall median survival (only 15 months) and 5-year survival rate. 2 Although many therapeutic strategies targeting have been developed, their application in the clinic for treatment GBM has been largely impeded due to the lack of safe and efficient drug delivery system that delivers drugs to tumor location. 3 Recent research findings suggest that, in various cancer types including GBM, human immune response has significant potential in promoting immune mediated tumor eradication and improving long term survival. 4 Recent studies have shown that anthracyclines, such as doxorubicin (DOX), not only induce apoptosis of tumor cells, but also

Background and Aims Transarterial chemoembolization (TACE) is a standard locoregional therapy for patients with hepatocellular carcinoma (HCC) patients with a variable overall response in efficacy. We aimed to identify key molecular signatures and related pathways leading to HCC resistance to TACE, with the hope of developing effective approaches in preselecting patients with survival benefit from TACE. Approach and Results Four independent HCC cohorts with 680 patients were used. MicroRNA (miRNA) transcriptome analysis in patients with HCC revealed a 41‐miRNA signature related to HCC recurrence after adjuvant TACE, and miR‐125b was the top reduced miRNA in patients with HCC recurrence. Consistently, patients with HCC with low miR‐125b expression in tumor had significantly shorter time to recurrence following adjuvant TACE in two independent cohorts. Loss of miR‐125b in HCC noticeably activated the hypoxia inducible factor 1 alpha subunit (HIF1α)/pAKT loop in vitro and in vivo. miR‐125b directly attenuated HIF1α translation through binding to HIF1A internal ribosome entry site region and targeting YB‐1, and blocked an autocrine HIF1α/platelet‐derived growth factor β (PDGFβ)/pAKT/HIF1α loop of HIF1α translation by targeting the PDGFβ receptor. The miR‐125b‐loss/HIF1α axis induced the expression of CD24 and erythropoietin (EPO) and enriched a TACE‐resistant CD24‐positive cancer stem cell population. Consistently, patients with high CD24 or EPO in HCC had poor prognosis following adjuvant TACE therapy. Additionally, in patients with HCC having TACE as their first‐line therapy, high EPO in blood before TACE was also noticeably related to poor response to TACE. Conclusions MiR‐125b loss activated the HIF1α/pAKT loop, contributing to HCC resistance to TACE and the key nodes in this axis hold the potential in assisting patients with HCC to choose TACE therapy.

Unfolded protein response (UPR) and endoplasmic reticulum (ER)-phagy are essential for cell homeostasis. Quantum dots (QDs), which have been widely used for biomedical applications, can accumulate in the kidney tissues and may cause renal dysfunction. However, the molecular mechanism of QDs-induced nephrotoxicity is still obscure. The present study was aimed to elucidate the role and mechanism of UPR and ER-phagy in QDs-induced nephrotoxicity. Herein, human embyronic kidney (HEK) cells were exposed to 15, 30, 45, and 60 nM cadmium telluride (CdTe)-QDs for 12 and 24 h. And CdTe-QDs (30-60 nM) inhibited the HEK cell viability. The clathrin-dependent endocytosis was determined as the main pathway of CdTe-QDs cellular uptake. Within cells, CdTe-QDs disrupted ER ultrastructure and induced UPR and FAM134B-dependent ER-phagy. Blocking UPR with inhibitors or siRNA rescued the FAM134B-dependent ER-phagy, which was triggered by CdTe-QDs. Moreover, suppression of UPR or FAM134B-dependent ER-phagy restored the cell vability. In vivo, mice were intravenously injected with 8 and 16 nmol/kg body weight CdTe-QDs for 24 h. Kidney was shown as one of highest distributed organs of CdTe-QDs, resulting in renal dysfunction, as well as UPR and FAM134B-dependent ER-phagy in it. Thus, for the first time, we demonstrated that ER-phagy can be triggered by nanomaterials both in vitro and in vivo. In addition, blocking of UPR and ER-phagy showed protective effects against CdTe-QDs-induced toxicity in kideny cells. Notably, a secreted alkaline phosphatase reporter gene system has been developed as a sensitive and rapid method for evaluating the ER quality under the exposure of nanomaterials.