Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC‐derived exosomes on angiogenesis. Exosomes derived from the NPC C666‐1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666‐1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose‐dependent manner. Subsequently, an iTRAQ‐based quantitative proteomics was used to identify the differentially expressed proteins in C666‐1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up‐ and down‐regulated (≥ 1.5‐fold variations) in C666‐1 exosomes compared to the normal counterparts, respectively. As expected, pro‐angiogenic proteins including intercellular adhesion molecule‐1 (ICAM‐1) and CD44 variant isoform 5 (CD44v5) are among the up‐regulated proteins, whereas angio‐suppressive protein, thrombospondin‐1 (TSP‐1) was down‐regulated in C666‐1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM‐1 and TSP‐1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes‐induced angiogenesis, which could potentially be developed as therapeutic targets in future.     

Exosome‐transmitted p120‐catenin suppresses hepatocellular carcinoma progression via STAT3 pathways

Hepatocellular carcinoma (HCC) is a fatal disease with increasing morbidity and poor prognosis due to surgical recurrence and metastasis. Moreover, the molecular mechanism of HCC progression remains unclear. Although the role of p120‐catenin (p120ctn) in liver cancer is well studied, the effects of secreted p120ctn transported by exosomes are less understood. Here, we show that p120ctn in exosomes secreted from liver cancer cells suppresses HCC cell proliferation and metastasis and expansion of liver cancer stem cells (CSCs). Mechanically, exosome p120ctn inhibits HCC cell progression via the STAT3 pathway, and the STAT3 inhibitor S3I‐201 abolishes the observed effects on growth, metastasis, and self‐renewal ability between exosome p120ctn‐treated HCC cells and control cells. Taken together, we propose that p120ctn‐containing exosomes derived from cancer cells inhibit the progression of liver cancer and may offer a new therapeutic strategy.     

Upregulation of caveolin‐1 and CD147 expression in nasopharyngeal carcinoma enhanced tumor cell migration and correlated with poor prognosis of the patients

Expression of caveolin‐1 (Cav‐1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) and their prognostic significance were analyzed in archive NPC samples. Cav‐1 and CD147 were overexpressed in 49.48% (96/194) and 59.39% (117/197) of NPC, respectively. Both Cav‐1 and CD147 expression levels correlated significantly with metastasis (p = 0.025 and 0.017, respectively) and a lower 5‐year survival rate (p = 0.02 and 0.0009, respectively). In addition, Cav‐1 expression levels correlated significantly with local recurrence (p = 0.038). Multivariate Cox regression analysis indicated that combination of high Cav‐1 and CD147 expression was a significant, independent prognosis predictor in patients with NPC (HR = 2.135; p = 0.006). Functional studies revealed that overexpression of Cav‐1 promoted secretion of MMP‐3 and MMP‐11 (active) proteins, as well as an increase in the migratory ability of CNE1 and CNE2 cells, while siRNA‐mediated silencing of Cav‐1 or CD147 led to reduced levels of MMP‐3 and MMP‐11(active) secretion, and reduced migration capacity of CNE1 and CNE2 cells. We observed a positive correlation between Cav‐1 and CD147 expression in NPC (ρ = 0.330, p = 0.000), CD147 protein levels were upregulated in Cav‐1 overexpressing CNE1 and CNE2 cells, whereas siRNA‐mediated silencing of Cav‐1 led to the downregulation of CD147 expression. Our results indicate that Cav‐1 and CD147 overexpression predict poor NPC prognosis and enhanced tumor cell migration, which is associated with MMP‐3 and MMP‐11 (active) secretion. © 2009 UICC     

Hypoxic nasopharyngeal carcinoma-derived exosomal miR-455 increases vascular permeability by targeting ZO-1 to promote metastasis

Nasopharyngeal carcinoma (NPC), the most frequent reason for treatment failure in head and neck tumors, has the greatest incidence of distant metastases. Increased vascular permeability facilitates metastasis. Exosomal microRNAs (miRNAs) have been implicated in the development of the premetastatic niche and are emerging as prospective biomarkers in cancer patients. We discovered that a higher level of miR-455 was connected to a larger propensity for NPC metastasis based on deep sequencing and RT-qPCR. We found that hypoxia promoted NPC exosomes release and increased miR-455 expression in a way that was hypoxia-inducible factor 1-alpha (HIF-1α) dependent. Exosomes from NPC cells with high levels of miR-455 were found to specifically target zonula occludens 1 (ZO-1), increasing the permeability of endothelial monolayers in vitro vascular permeability and transendothelial invasion experiments. Additional in vivo studies showed that zebrafish with sustained miR-455-overexpressing NPC cell xenografts displayed increased tumor cell mass throughout the body. In vivo, zebrafish vascular tight junction integrity was disrupted by exosomes produced by NPC cells with elevated miR-455 expression. Mice-bearing xenografts further supported the finding that exosomes containing miR-455 might reduce ZO-1 expression in addition to promote NPC cell growth. These findings suggest that in a hypoxic microenvironment, exosomal miR-455 released by NPC cells enhances vascular permeability and promotes metastasis by targeting ZO-1. The HIF-1α-miR-455-ZO-1 signaling pathway may be a promising predictor and potential therapeutic target for NPC with metastasis.     

Exosomes containing ErbB2/CRK induce vascular growth in premetastatic niches and promote metastasis of bladder cancer

Locally advanced and metastatic invasive bladder cancer (BC) has a poor prognosis, and no advanced therapies beyond cisplatin‐based combination chemotherapy have been developed. Therefore, it is an urgent issue to elucidate the underlying mechanisms of tumor progression and metastasis of invasive BC for the development of new therapeutic strategies. Here, we clarified a novel role of exosomes containing ErbB2 and CRK in a formation of premetastatic niches and subsequent metastases. CRK adaptors were overexpressed in invasive UM‐UC‐3 BC cells. In an orthotopic xenograft model, metastases to lung, liver, and bone of UM‐UC‐3 cells were completely abolished by CRK elimination. Mass spectrometry analysis identified that ErbB2 was contained in UM‐UC‐3‐derived exosomes in a CRK‐dependent manner; the exosomes significantly increased proliferation and invasion properties of low‐grade 5637 BC cells and HUVECs through FAK and PI3K/AKT signaling pathways. In athymic mice educated with UM‐UC‐3‐derived exosomes, i.v. implanted UM‐UC‐3 cells were trapped with surrounding PKH67‐labeled exosomes in lung and led to development of lung metastasis with disordered vascular proliferation. In contrast, exosomes derived from CRK‐depleted BC cells failed to induce these malignant features. Taken together, we showed that CRK adaptors elevated the expression of ErbB2/3 in BC cells, and these tyrosine kinase/adaptor units were transferred from host BC cells to metastatic recipient cells by exosomes, leading to vascular leakiness and proliferation and contributing to the formation of distant metastasis. Thus, CRK intervention with ErbB2/3 blockade might be a potent therapeutic strategy for patients with ErbB2 overexpressing advanced and metastatic BC.     

Omental cancer-associated fibroblast-derived exosomes with low microRNA-29c-3p promote ovarian cancer peritoneal metastasis

Ovarian cancer (OC) is characterized by frequent widespread peritoneal metastasis. Cancer-associated fibroblasts (CAFs) represent a critical stromal component of metastatic niche and promote omentum metastasis in OC patients. However, the role of exosomes derived from omental CAFs in metastasis remains unclear. We isolated exosomes from primary omental normal fibroblasts (NFs) and CAFs from OC patients (NF-Exo and CAF-Exo, respectively) and assessed their effect on metastasis. In mice bearing orthotopic OC xenografts, CAF-Exo treatment led to more rapid intraperitoneal tumor dissemination and shorter animal survival. Similar results were observed in mice undergoing intraperitoneal injection of tumor cells. Among the miRNAs downregulated in CAF-Exo, miR-29c-3p in OC tissues was associated with metastasis and survival in patients. Moreover, increasing miR-29c-3p in CAF-Exo significantly weakened the metastasis-promoting effect of CAF-Exo. Based on RNA sequencing, expression assays, and luciferase assays, matrix metalloproteinase 2 (MMP2) was identified as a direct target of miR-29c-3p. These results verify the significant contribution of exosomes from omental CAFs to OC peritoneal metastasis, which could be partially due to the relief of MMP2 expression inhibition mediated by low exosomal miR-29c-3p.     

Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer

Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a “stemness and metastatic” signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.     

Exosomes secreted by prostate cancer cells under hypoxia promote matrix metalloproteinases activity at pre-metastatic niches

Approximately, 30 000 men die from prostate cancer (PCa) every year in the United States, mainly due to the metastasis. Thus, the key events associated with PCa metastasis are under rigorous investigation, with recent studies showing that preparation of pre-metastatic niches (PMN) in distant organs is an important step. However, the molecular basis for PMN preparation is still unclear. Hypoxia in primary tumors promotes aggressiveness; however, its precise role in metastasis is not clear. We recently reported that exosomes secreted by PCa cells under hypoxia promote stemness and invasiveness in naïve PCa cells; however, whether these extracellular vesicles also influence PMN remains unknown. In the present study, we isolated exosomes from human PCa PC3 cells under normoxic (21% O₂, exosomes secreted under normoxic condition [Exo^Normoxic]) and hypoxic (1% O₂, exosomes secreted under hypoxic condition [Exo^Hypoxic]) conditions, and characterized their effect (10 µg exosomes, intraperitoneal (IP) treatment every 48 hours for 4 weeks) on key biomarkers associated with PMN in nude mice. Whole animal fluorescence imaging showed that Exo^Hypoxic treatment promotes matrix metalloproteinases (MMPs) activity in several putative metastatic sites. Histological studies confirmed that Exo^Hypoxic treatment enhanced the level of MMP2, MMP9, and extracellular matrix proteins (fibronectin and collagen) as well as increased the number of CD11b+ cells at selective PMN sites. Furthermore, proteomic profiling of exosomes by liquid chromatography/mass spectrometry identified cargo proteins in Exo^Normoxic and Exo^Hypoxic as well as distinct canonical pathways targeted by them. These results suggest that exosomes secreted by PCa cells under hypoxia plausibly remodel distant PMN, and thus, could be a potential target to control metastatic PCa.     

Complex roles of tissue inhibitors of metalloproteinases in cancer

Matrix metalloproteinases (MMPs) is tightly associated with extracellular matrix (ECM) turnover, which plays a very active role in tumor invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) plays a critical role in the homeostasis of ECM by regulating the activity of MMPs. TIMPs are well-known for their ability to inhibit MMP activity thereby inhibiting tumor growth and metastasis. However, many evidences suggest that TIMPs are multifunctional proteins, which regulate cell proliferation, apoptosis, proMMP-2 activation, and angiogenesis. These effects may be through MMP-dependent or MMP-independent pathways. Recent data indicate that TIMPs have many paradoxical roles in tumorigenesis. In particular, both inhibitory effect and stimulatory effect on tumorigenesis have been demonstrated in many animal models in which TIMPs were overexpressed in cancer cells or in mice. Elevated TIMP levels are reported in association with cancer progression and identified as poor prognostic indicators in several human tumor types. Herein, we review the complex roles of TIMPs in cancer growth and metastasis.     

ING2 is upregulated in colon cancer and increases invasion by enhanced MMP13 expression

Inhibitor of growth 2 (ING2) is associated with chromatin remodeling and regulation of gene expression by binding to a methylated histone H3K4 residue and recruiting HDAC complexes to the region. The aim of our study is to investigate the regulation of ING2 expression and the clinical significance of upregulated ING2 in colon cancer. Here, we show that the ING2 mRNA level in colon cancer tissue increased to more than twice than that in normal mucosa in the 45% of colorectal cancer cases that we examined. A putative NF‐κB binding site was found in the ING2 promoter region. We confirmed that NF‐κB could bind to the ING2 promoter by EMSA and luciferase assays. Subsequent microarray analyses revealed that ING2 upregulates expression of matrix metalloproteinase 13 (MMP13), which enhances cancer invasion and metastasis. ING2 regulation of MMP13 expression was confirmed in both ING2 overexpression and knock down experiments. MMP13 expression was further induced by coexpression of ING2 with HDAC1 or with mSin3A, suggesting that the ING2‐HDAC1‐mSin3A complex members regulates expression of MMP13. In vitro invasion assay was performed to determine functional significance of ING2 upregulation. ING2 overexpressed cells exhibited greater invasive potential. Taken together, upregulation of ING2 was associated with colon cancer and MMP13‐dependent cellular invasion, indicating that ING2 expression might be involved with cancer invasion and metastasis. Published 2009 UICC.     

间充质干细胞来源外泌体在恶性肿瘤中的研究进展

间充质干细胞（mesenchymal stem cells,MSCs）是存在于各种组织中的多能基质细胞。外泌体为细胞间的通讯载体,能在细胞间传递脂质、核酸以及蛋白质等生物活性分子。MSCs分泌的外泌体（mesenchymal stem cell-derived exosomes,MSC-EXO）为肿瘤微环境（tumor microenvironment,TME）的主要组成部分,并且在肿瘤的发生发展、血管生成及转移过程中发挥重要作用。本文旨在对MSCs来源的外泌体在癌症研究及其对肿瘤的作用机制予以综述,为适当利用修饰的MSC-EXO作为肿瘤治疗的策略提供新思路。      

The versatile role of exosomes in cancer progression: diagnostic and therapeutic implications

Background

Recent advances in cancer biology have highlighted the relevance of exosomes and nanovesicles as carriers of genetic and biological messages between cancer cells and their immediate and/or distant environments. It has been found that these molecular cues may play significant roles in cancer progression and metastasis. Cancer cells secrete exosomes containing diverse molecules that can be transferred to recipient cells and/or vice versa to induce a plethora of biological processes, including angiogenesis, metastasis formation, therapeutic resistance, epithelial-mesenchymal transition and epigenetic/stemness (re)programming. While exosomes interact with cells within the tumour microenvironment to promote tumour growth, these vesicles can also facilitate the process of distant metastasis by mediating the formation of pre-metastatic niches. Next to their tumour promoting effects, exosomes have been found to serve as potential tools for cancer diagnosis and therapy. The ease of isolating exosomes and their content from different body fluids has led to the identification of diagnostic and prognostic biomarker signatures, as well as to predictive biomarker signatures for therapeutic responses. Exosomes can also be used as cargos to deliver therapeutic anti-cancer drugs, and they can be engineered to serve as vaccines for immunotherapy. Additionally, it has been found that inhibition of exosome secretion, and thus the transfer of oncogenic molecules, holds promise for inhibiting tumour growth. Here we provide recent information on the diverse roles of exosomes in various cellular and systemic processes governing cancer progression, and discuss novel strategies to halt this progression using exosome-based targeted therapies and methods to inhibit exosome secretion and the transfer of pro-tumorigenic molecules.

Conclusions

This review highlights the important role of exosomes in cancer progression and its implications for (non-invasive) diagnostics and the development of novel therapeutic strategies, as well as its current and future applications in clinical trials.

     

Formin-like 2 promotes angiogenesis and metastasis of colorectal cancer by regulating the EGFL6/CKAP4/ERK axis

Increasing evidence indicates that angiogenesis plays a pivotal role in tumor progression. Formin-like 2 (FMNL2) is well-known for promoting metastasis; however, the molecular mechanisms by which FMNL2 promotes angiogenesis in colorectal cancer (CRC) remain unclear. Here, we found that FMNL2 promotes angiogenesis and metastasis of CRC in vitro and in vivo. The GDB/FH3 domain of FMNL2 directly interacts with epidermal growth factor-like protein 6 (EGFL6). Formin-like 2 promotes EGFL6 paracrine signaling by exosomes to regulate angiogenesis in CRC. Cytoskeleton associated protein 4 (CKAP4) is a downstream target of EGFL6 and is involved in CRC angiogenesis. Epidermal growth factor-like protein 6 binds to the N-terminus of CKAP4 to promote the migration of HUVECs by activating the ERK/MMP pathway. These findings suggest that FMNL2 promotes the migration of HUVECs and enhances angiogenesis and tumorigenesis in CRC by regulating the EGFL6/CKAP4/ERK axis. Therefore, the EGFL6/CKAP4/ERK axis could be a candidate therapeutic target for CRC treatment.     

Role of stem cell derived exosomes in tumor biology

Exosomes are nano‐scale messengers loaded with bio‐molecular cargo of RNA, DNA, and Proteins. As a master regulator of cellular signaling, stem cell (both normal, and cancer stem cells) secreted exosome orchestrate various autocrine and paracrine functions which alter tumor micro‐environment, growth and progression. Exosomes secreted by one of the two important stem cell phenotypes in cancers a) Mesenchymal stem cells, and b) Cancer stem cells not only promote cancerous growth but also impart therapy resistance in cancer cells. In tumors, normal or mesenchymal stem cell (MSCs) derived exosomes (MSC‐exo) modulate tumor hallmarks by delivering unique miRNA species to neighboring cells and help in tumor progression. Apart from regulating tumor cell fate, MSC‐exo are also capable of inducing physiological processes, for example, angiogenesis, metastasis and so forth. Similarly, cancer stem cells (CSCs) derived exosomes (CSC‐exo) contain stemness‐specific proteins, self‐renewal promoting regulatory miRNAs, and survival factors. CSC‐exo specific cargo maintains tumor heterogeneity and alters tumor progression. In this review we critically discuss the importance of stem cell specific exosomes in tumor cell signaling pathways with their role in tumor biology.     

Exosomes Derived from Irradiated Esophageal Carcinoma-Infiltrating T Cells Promote Metastasis by Inducing the Epithelial–Mesenchymal Transition in Esophageal Cancer Cells

Exosomes are nanovesicles derived from tumor and normal cells that are detectable in human biological fluids, such as plasma, and cell culture supernatants. The function of exosome secretion from “normal” cells is unclear. Although numerous studies have investigated exosomes derived from hematopoietic cells, little is known regarding exosomes fromT cells, even though these cells play significant roles in innate and acquired immunity. A CCK-8 assay was used to examine the ability of exosomes to inhibit TE13 cell proliferation. In vitro invasion and wound healing assays were conducted to explore the effects of exosomes on TE13 cell migration and invasion. A Western blottinganalys is was performed to investigate the effects of exosomes on the expression of the EMT-related moleculesβ-catenin, NF-κB and snail. This study aimed to investigate the effects of exosomes from irradiated T cells on the human esophageal squamous cell carcinoma (ESCC) cell line TE13 and revealed that exosomes inhibit the proliferation but promote the metastasis of TE13 cells in a dose-and time-dependent manner. Furthermore, exosomes significantly increased the expression of β-catenin, NF-κB and snail in TE13 cells. The results of this study suggest an important role for T cell-derived exosomes in the progression of esophageal carcinoma: T cell-derived exosomes promote esophageal cancer metastasis, likely by promoting the EMT through the upregulation of β-catenin and the NF-κB/snail pathway. Moreover, this study supports the use of exosomes as a nearly perfect example of biomimetic nanovesicles that could be utilized in future therapeutic strategies against various diseases, including cancer.     

Macrophage migration inhibitory factor promotes the invasion and metastasis of oral squamous cell carcinoma through matrix metalloprotein‐2/9

Macrophage migration inhibitory factor (MIF) has been shown to closely associate with the malignant progression of a variety of human carcinomas. However, the role and its underlying molecular mechanisms of MIF in the invasion and metastasis of oral squamous cell carcinoma (OSCC) still remains unclear. Here, we found that MIF silencing reduced the cell proliferation, migration, and invasion, as well as matrix metalloprotein‐2 (MMP‐2) and MMP‐9 in OSCC cells. Overexpression of MMP‐2 or MMP‐9 restored the migration and invasion of MIF‐knockdown cells, indicating that MMP‐2 and MMP‐9 are downstream targets of MIF. In the xenograft model, MIF silencing inhibited tumor growth and in lymph metastasis model, MIF silencing reduced tumor metastasis. More importantly, immunohistochemistry staining in a tissue microarray (TMA) demonstrated that MIF expression was positively correlated with clinic stage, recurrence, metastasis, and poor prognosis of patients with OSCC as well as with the levels of MMP‐2 or MMP‐9 in TMA. Therefore, our findings suggest that MIF may promote the invasion and metastasis of OSCC through the activation of MMP‐2 and MMP‐9 and prompt further investigation into the therapeutic value of MIF for OSCC treatment.     

RNAi targeting urokinase‐type plasminogen activator receptor inhibits metastasis and progression of oral squamous cell carcinoma in vivo

It has been admitted that urokinase‐type plasminogen activator receptor (u‐PAR) is overexpressed in many human malignant tumors including oral squamous cell carcinoma (OSCC) and plays an important role in a variety of cancer key cellular events as a versatile signaling orchestrator. In our study, a retroviral vector expressing u‐PAR‐specific siRNA was injected into OSCC xenografts of nude mice to observe its inhibitory effects on OSCC. Our data demonstrate that siRNA targeting u‐PAR markedly suppressed tumor growth, reduced the expression of proliferation‐related gene, Ki‐67 and increased cell apoptosis, accompanying with the efficient and specific inhibition of endogenous u‐PAR expression in OSCC. More importantly, the mRNA and protein expression of MMP‐2, MMP‐9, VEGF‐C, VEGF‐D and VEGFR‐3 that are intimately involved in oral cancer invasion and metastasis, was simultaneously downregulated significantly as determined by quantitative real‐time RT‐PCR, Western blot and immunohistochemistry; and Gelatin and fibrin zymography showed that MMP‐9, MMP‐2 and u‐PA enzymatic activities were significantly reduced in u‐PAR‐specific siRNA group, compared to those in control groups. In addition, the expression of MDR‐1 gene related to drug resistance was obviously inhibited by silencing of u‐PAR. These findings suggest that RNAi targeting u‐PAR could effectively inhibit the metastasis and progression of OSCC in vivo. Thus, it may be used as a potent and specific therapy for oral cancer, especially in inhibiting and preventing cancer cell invasion and metastasis. © 2009 UICC