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.     

p120-catenin亚型的表达减弱与肺癌的淋巴结转移明显相关

背景与目的 已有的研究表明,p120ctn(p120 catenin)在细胞粘附中起重要作用,与肿瘤发生关系密切.本研究探讨p120ctn哑型在肺鳞癌、腺癌中的表达及其与各临床病理因素的关系.方法应用RT-PCR方法检测肺鳞癌、腺痛及对应癌旁肺组织中p120ctn各亚型的表达情况,同时应用免疫荧光、Western-blot和RT-PCR方法检测了p120ctn各业型在肺癌PG细胞系BE1和LH7中的表达.结果 p120ctn的总mRNA及其亚型1和亚型3的mRNA在癌旁肺组织中的表达量要明显高于肺鳞、腺痛组织中的表达量(P<0.001,P<0.001,P=0.001).且亚型1 mRNA的表达缺失与淋巴结转移呈负相关(P=0.01),而亚型3 mRNA的表达缺失与癌组织的淋巴结转移呈正相关(P=0.029).高转移能力的BE1细胞中p120ctn亚型1和亚型3的表达减弱比LH7更为明显.结论 p120ctn亚型1和3 mRNA的表达减弱或缺失是肺癌中普遍存在的现象,且这种现象与淋巴结的转移有密切关系.     

Fatty liver creates a pro‐metastatic microenvironment for hepatocellular carcinoma through activation of hepatic stellate cells

Fatty liver (FL) is associated with development of hepatocellular carcinoma (HCC). However, whether FL itself promotes the progression of HCC is unclear. We recently found that hepatic stellate cells (HSCs) were prominently activated in the steatotic liver. Here, we investigated whether steatotic livers promote HCC progression and whether HSCs of steatotic liver are associated with HCC progression. We implanted rat HCC cells into diet‐induced steatotic livers in rats via portal vein injection. Thereafter, HSCs and HCC cells were co‐implanted subcutaneously into nude rats. Migration and proliferation of HCC cells were measured, and activation of ERK and Akt in these cells was determined by western blotting. Chemokines secreted from HSCs and HCC cells were also evaluated by ELISA. Steatotic livers significantly promoted HCC metastasis compared with non‐steatotic livers. Additionally, co‐implantation of HCC cells with HSCs from steatotic livers produced significantly larger tumors in recipient rats as compared to those induced by HCC cells co‐implanted with HSCs from normal livers (NLs). HSCs isolated from steatotic livers, compared with HSCs isolated from NLs, secreted greater amounts of interleukin‐1α, vascular endothelial growth factor, and transforming growth factor‐β. These cytokines may enhance the proliferation and migration of HCC cells by increasing the phosphorylation of ERK and Akt in HCC cells. Moreover, we noted that the Rho‐kinase inhibitor deactivated activated HSCs and attenuated HCC progression. In conclusion, the rat steatotic liver microenvironment favors HCC metastasis, and this effect appears to be promoted by activated HSCs in the steatotic liver.     

Exosomes in cancer development and clinical applications

Exosomes participate in cancer progression and metastasis by transferring bioactive molecules between cancer and various cells in the local and distant microenvironments. Such intercellular cross‐talk results in changes in multiple cellular and biological functions in recipient cells. Several hallmarks of cancer have reportedly been impacted by this exosome‐mediated cell‐to‐cell communication, including modulating immune responses, reprogramming stromal cells, remodeling the architecture of the extracellular matrix, or even endowing cancer cells with characteristics of drug resistance. Selectively, loading specific oncogenic molecules into exosomes highlights exosomes as potential diagnostic biomarkers as well as therapeutic targets. In addition, exosome‐based drug delivery strategies in preclinical and clinical trials have been shown to dramatically decrease cancer development. In the present review, we summarize the significant aspects of exosomes in cancer development that can provide novel strategies for potential clinical applications.     

Wnt5b‐associated exosomes promote cancer cell migration and proliferation

Wnt5b is a member of the same family of proteins as Wnt5a, the overexpression of which is associated with cancer aggressiveness. Wnt5b is also suggested to be involved in cancer progression, however, details remain unclarified. We analyzed the biochemical properties of purified Wnt5b and the mode of secretion of Wnt5b by cancer cells. Wnt5b was glycosylated at three asparagine residues and lipidated at one serine residue, and these post‐translational modifications of Wnt5b were essential for secretion. Purified Wnt5b showed Dvl2 phosphorylation and Rac activation abilities to a similar extent as Wnt5a. In cultured‐cell conditioned medium, Wnt5b was detected in supernatant or precipitation fractions that were separated by centrifugation at 100 000 g. In PANC‐1 pancreatic cancer cells, 55% of secreted endogenous Wnt5b was associated with exosomes. Exosomes from wild‐type PANC‐1 cells, but not those from Wnt5b‐knockout PANC‐1 cells, activated Wnt5b signaling in CHO cells and stimulated migration and proliferation of A549 lung adenocarcinoma cells, suggesting that endogenous, Wnt5b‐associated exosomes are active. The exosomes were taken up by CHO cells and immunoelectron microscopy revealed that Wnt5b is indeed associated with exosomes. In Caco‐2 colon cancer cells, most Wnt5b was recovered in precipitation fractions when Wnt5b was ectopically expressed (Caco‐2/Wnt5b cells). Knockdown of TSG101, an exosome marker, decreased the secretion of Wnt5b‐associated exosomes from Caco‐2/Wnt5b cells and inhibited Wnt5b‐dependent cell proliferation. Exosomes secreted from Caco‐2/Wnt5b cells stimulated migration and proliferation of A549 cells. These results suggest that Wnt5b‐associated exosomes promote cancer cell migration and proliferation in a paracrine manner.     

Clinical impact of serum exosomal microRNA‐21 as a clinical biomarker in human esophageal squamous cell carcinoma

BACKGROUND:

Exosomes are 40‐nm to 100‐nm membrane vesicles that are secreted by various cells, and they play a major role in cell‐cell communication. The objective of this study was to clarify the significance of the levels of microRNA in exosomes extracted from the sera of patients with esophageal squamous cell cancer (ESCC).

METHODS:

The authors isolated exosomes in serum samples from patients who had ESCC and from patients who had benign diseases without systemic inflammation. Total RNA was purified from the exosomes, and expression levels of microRNA‐21 (miR‐21) were analyzed by quantitative real‐time polymerase chain reaction.

RESULTS:

Serum exosomes from patients with ESCC induced the proliferation of ESCC cells in vitro. The expression levels of exosomal miR‐21 were significantly higher in patients with ESCC than those with benign diseases with and without (C‐reactive protein <0.3 mg/dL) systemic inflammation. MiR‐21 was not detected in serum that remained after exosome extraction. Exosomal miR‐21 expression was correlated with advanced tumor classification, positive lymph node status, and the presence of metastasis with inflammation or and clinical stage without inflammation (C‐reactive protein <0.3 mg/dL).

CONCLUSIONS:

The current results confirmed that exosomal miR‐21 expression is up‐regulated in serum from patients with ESCC versus serum from patients who have benign diseases without systemic inflammation. Exosomal miR‐21 was positively correlated with tumor progression and aggressiveness, suggesting that it may be a useful target for cancer therapy. Cancer 2013. © 2012 American Cancer Society.     

p120ctn亚型在肺鳞癌、腺癌中表达及意义

背景与目的已有的研究显示:p120ctn(p120 catenin)与肿瘤的发生密切相关,在保持细胞黏附中起着重要作用,本研究通过观察p120ctn各亚型在肺鳞癌、腺癌和相应的癌旁肺组织中蛋白表达量的变化,探讨p120ctn亚型与各临床病理因素的关系。方法应用免疫荧光和Western Blot方法检测肺鳞癌、腺癌及对应癌旁正常肺组织中p120ctn各亚型的表达情况。结果与正常肺组织相比,p120ctn在肺癌细胞膜表达减弱,呈现胞膜荧光不连续或缺失,胞浆内存在p120ctn的蓄积。正常肺组织中p120ctn总蛋白量明显高于肺癌组织(P<0.001)。p120ctn蛋白以亚型1(120KD)和亚型3(100KD)表达为主,而在肺癌组织则出现1、3亚型表达缺失或减弱(P=0.001,P<0.001)。并且亚型3的这种缺失或减弱的现象与肺癌的淋巴结转移负相关(P=0.005)。结论p120ctn亚型1和3的蛋白表达的减弱或缺失是肺癌的普遍现象,且这种现象与淋巴结的转移相关。     

Altered localization of p120 catenin during epithelial to mesenchymal transition of colon carcinoma is prognostic for aggressive disease

We examined the expression and localization of p120 catenin (p120ctn) as a consequence of the epithelial to mesenchymal transition (EMT) of highly differentiated colon carcinoma cells (LIM1863 cells). This unique line grows in suspension as spheroids and undergoes an EMT within 24 hours following stimulation with transforming growth factor-beta and tumor necrosis factor-alpha. Although p120ctn expression remains stable during the EMT, its localization shifts from cell-cell junctions to the cytoplasm. Interestingly, a marked decrease in RhoA activation coincident with E-cadherin loss occurs during the EMT and correlates with the formation of a p120ctn/RhoA complex. Use of RNA interference showed that p120ctn reduction results in increased RhoA activity and a significant decrease in the motility of post-EMT cells. To determine the relevance of these findings to colorectal cancer progression, we assessed p120ctn expression by immunohistochemistry in 557 primary tumors. Of note, we observed that 53% of tumors presented cytoplasmic staining for p120ctn, and statistical analysis revealed that this localization is predictive of poor patient outcome. Cytoplasmic p120ctn correlated with later-stage tumors, significantly reduced 5- and 10-year survival times and a greater propensity for metastasis to lymph nodes compared with junctional p120ctn. We also confirmed that altered localization of p120ctn corresponded with loss or cytoplasmic localization of E-cadherin. These alterations in E-cadherin are also associated with a significant reduction in patient survival time and an increase in tumor stage and lymph node metastasis. These data provide a compelling argument for the importance of both p120ctn and the EMT itself in the progression of colorectal carcinoma.     

MicroRNA‐204‐5p: A novel candidate urinary biomarker of Xp11.2 translocation renal cell carcinoma

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early‐stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC‐TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)‐204‐5p levels in urinary exosomes of 40‐week‐old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA‐204‐5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR‐204‐5p‐containing exosomes. Notably, we also observed increased miR‐204‐5p levels in urinary exosomes in 20‐week‐old renal PRCC‐TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40‐week‐old Tg mice, suggesting that miR‐204‐5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR‐204‐5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC‐TFE3 fusion gene. These findings suggest that miR‐204‐5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC.     

Induction of myeloid‐derived suppressor cells by tumor exosomes

Myeloid‐derived suppressor cells (MDSCs) promote tumor progression. The mechanisms of MDSC development during tumor growth remain unknown. Tumor exosomes (T‐exosomes) have been implicated to play a role in immune regulation, however the role of exosomes in the induction of MDSCs is unclear. Our previous work demonstrated that exosomes isolated from tumor cells are taken up by bone marrow myeloid cells. Here, we extend those findings showing that exosomes isolated from T‐exosomes switch the differentiation pathway of these myeloid cells to the MDSC pathway (CD11b⁺Gr‐1⁺). The resulting cells exhibit MDSC phenotypic and functional characteristics including promotion of tumor growth. Furthermore, we demonstrated that in vivo MDSC mediated promotion of tumor progression is dependent on T‐exosome prostaglandin E2 (PGE2) and TGF‐β molecules. T‐exosomes can induce the accumulation of MDSCs expressing Cox2, IL‐6, VEGF, and arginase‐1. Antibodies against exosomal PGE2 and TGF‐β block the activity of these exosomes on MDSC induction and therefore attenuate MDSC‐mediated tumor‐promoting ability. Exosomal PGE2 and TGF‐β are enriched in T‐exosomes when compared with exosomes isolated from the supernatants of cultured tumor cells (C‐exosomes). The tumor microenvironment has an effect on the potency of T‐exosome mediated induction of MDSCs by regulating the sorting and the amount of exosomal PGE2 and TGF‐β available. Together, these findings lend themselves to developing specific targetable therapeutic strategies to reduce or eliminate MDSC‐induced immunosuppression and hence enhance host antitumor immunotherapy efficacy. © 2008 Wiley‐Liss, Inc.     

MicroRNA‐493‐5p‐mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

Primary hepatic tumors mainly include hepatocellular carcinoma (HCC), which is one of the most frequent causes of cancer‐related deaths worldwide. Thus far, HCC prognosis has remained extremely poor given the lack of effective treatments. Numerous studies have described the roles played by microRNAs (miRNAs) in cancer progression and the potential of these small noncoding RNAs for diagnostic or therapeutic applications. The current consensus supports the idea that direct repression of a wide range of oncogenes by a single key miRNA could critically affect the malignant properties of cancer cells in a synergistic manner. In this study, we aimed to investigate the oncogenes controlled by miR‐493‐5p, a major tumor suppressor miRNA that inactivates miR‐483‐3p oncomir in hepatic cancer cells. Using global gene expression analysis, we highlighted a set of candidate genes potentially regulated by miR‐493‐5p. In particular, the canonical MYCN protooncogene (MYCN) appeared to be an attractive target of miR‐493‐5p given its significant inhibition through 3′‐UTR targeting in miR‐493‐5p‐rescued HCC cells. We showed that MYCN was overexpressed in liver cancer cell lines and clinical samples from HCC patients. Notably, MYCN expression levels were inversely correlated with miR‐493‐5p in tumor tissues. We confirmed that MYCN knockdown mimicked the anticancer effect of miR‐493‐5p by inhibiting HCC cell growth and invasion, whereas MYCN rescue hindered miR‐493‐5p activity. In summary, miR‐493‐5p is a pivotal miRNA that modulates various oncogenes after its reexpression in liver cancer cells, suggesting that tumor suppressor miRNAs with a large spectrum of action could provide valuable tools for miRNA replacement therapies.     

The bone marrow microenvironment enhances multiple myeloma progression by exosome-mediated activation of myeloid-derived suppressor cells

Exosomes, extracellular nanovesicles secreted by various cell types, modulate the bone marrow (BM) microenvironment by regulating angiogenesis, cytokine release, immune response, inflammation, and metastasis. Interactions between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells play crucial roles in MM development. We previously reported that BMSC-derived exosomes directly promote MM cell growth, whereas the other possible mechanisms for supporting MM progression by these exosomes are still not clear. Here, we investigated the effect of BMSC-derived exosomes on the MM BM cells with specific emphasis on myeloid-derived suppressor cells (MDSCs). BMSC-derived exosomes were able to be taken up by MM MDSCs and induced their expansion in vitro. Moreover, these exosomes directly induced the survival of MDSCs through activating STAT3 and STAT1 pathways and increasing the anti-apoptotic proteins Bcl-xL and Mcl-1. Inhibition of these pathways blocked the enhancement of MDSC survival. Furthermore, these exosomes increased the nitric oxide release from MM MDSCs and enhanced their suppressive activity on T cells. Taken together, our results demonstrate that BMSC-derived exosomes activate MDSCs in the BM through STAT3 and STAT1 pathways, leading to increased immunosuppression which favors MM progression.     

p120ctn isoform 1 expression significantly correlates with abnormal expression of E-cadherin and poor survival of lung cancer patients

Different p120ctn isoforms exert different, even opposing, effects on tumor cell growth depending on the level of E-cadherin expression, but the impact on clinicopathological parameters of lung cancer patients is not clear. Herein, we investigate the correlation between pan-p120ctn, p120ctn isoform 1, and E-cadherin expression and clinicopathological parameters, especially prognosis, of lung cancer patients. Immunohistochemistry on 20 specimens of normal bronchial epthelium revealed that, p120ctn isoform 1 was not expressed at the membrane; only weak cytoplasmic expression was seen. In contrast, both pan-p120ctn and E-cadherin were expressed clearly on the cell membrane or in the cytoplasmic peri-membrane region. However, in squamous cell lung cancer or lung adenocarcinomas, p120ctn isoform 1 over-expressed in the cytoplasm accompany with the abnormal pan-p120ctn and E-cadherin cytoplasm expression. p120ctn isoform 1 over-expression correlated positively with lymph node metastasis, poor differentiation, histological type, and high TNM stage. Cytoplasmic p120ctn isoform 1 expression in metastatic nodules was always higher than in the primary tumor. While the mean survival times of patients with normal p120 ctn isoform 1 or pan-p120ctn expression differed significantly, the mean survival times of patients with abnormal expression were similar. Lymph node metastasis, TNM stage, abnormal pan-p120ctn expression, and p120ctn isoform 1 over-expression were all independent factors affecting the prognosis of lung cancer patients. Over-expression of p120ctn isoform 1 positively correlated with poor prognosis of lung cancer patients, and therefore may be a useful marker of lung cancer patient survival.     

Matrix metalloproteinase 13‐containing exosomes promote nasopharyngeal carcinoma metastasis

Nasopharyngeal cancer (NPC) is an endemic type of head and neck cancer with a high rate of cervical lymph node metastasis. Metastasis is the major cause of death in NPC patients. Increasing evidence indicates that exosomes play a pivotal role in promoting cancer metastasis by enhancing angiogenesis and ECM degradation. Matrix metalloproteinase 13 is an important kind of matrix proteinase that is often overexpressed in various tumors and increases the risk of metastasis. However, little is known about the potential role of MMP13‐containing exosomes in NPC. In this study, we found that MMP13 was overexpressed in NPC cells and exosomes purified from conditioned medium (CM) as well as NPC patients’ plasma. Transwell analysis revealed that MMP13‐containing exosomes facilitated the metastasis of NPC cells. Furthermore, siRNA inhibited the effect of MMP13‐containing exosomes on tumor cells metastasis as well as angiogenesis. The current findings provided novel insight into the vital role of MMP13‐containing exosomes in NPC progression which might offer unique insights for potential therapeutic strategies for NPC progressions.     

Increased T‐helper 17 cell differentiation mediated by exosome‐mediated microRNA‐451 redistribution in gastric cancer infiltrated T cells

MicroRNA (miR)‐451 is a cell metabolism‐related miRNA that can mediate cell energy‐consuming models by several targets. As miR‐451 can promote mechanistic target of rapamycin (mTOR) activity, and increased mTOR activity is related to increased differentiation of T‐helper 17 (Th17) cells, we sought to investigate whether miR‐451 can redistribute from cancer cells to infiltrated T cells and enhance the distribution of Th17 cells through mTOR. Real‐time PCR was used for detecting expression of miR‐451 in gastric cancer, tumor infiltrated T cells and exosomes, and distribution of Th17 was evaluated by both flow cytometry and immunohistochemistry (IHC). Immunofluorescence staining was used in monitoring the exosome‐enveloped miR‐451 from cancer cells to T cells with different treatments, and signaling pathway change was analyzed by western blot. miR‐451 decreased significantly in gastric cancer (GC) tissues but increased in infiltrated T cells and exosomes; tumor miR‐451 was negatively related to infiltrated T cells and exosome miR‐451. Exosome miR‐451 can not only serve as an indicator for poor prognosis of post‐operation GC patients but is also related to increased Th17 distribution in gastric cancer. miR‐451 can redistribute from cancer cells to T cells with low glucose treatment. Decreased 5′ AMP‐activated protein kinase (AMPK) and increased mTOR activity was investigated in miR‐451 redistributed T cells and the Th17 polarized differentiation of these T cells were also increased. Exosome miR‐451 derived from tumor tissues can serve as an indicator for poor prognosis and redistribution of miR‐451 from cancer cells to infiltrated T cells in low glucose treatment can enhance Th17 differentiation by enhancing mTOR activity.     

外泌体在癌症诊治中的作用与机制

外泌体是一种新型的癌症生物标志物，它由所有体液中各种活细胞分泌的双层纳米囊泡构成，含有丰富的蛋白质、DNA、mRNA和非编码RNA。目前外泌体被认为是细胞间通讯的另一种机制，参与细胞间交换蛋白质、脂质和遗传物质。越来越多的研究表明，外泌体在肿瘤的发生、生长、进展、转移、耐药性和免疫逃逸中发挥重要作用。在本文中，我们根据外泌体生物学的最新进展，详细阐述了外泌体影响肿瘤之间通信的具体机制，并报道了外泌体可能成为癌症诊断中有前途的生物标志物，并代表癌症治疗的新靶点。