Exosome-mediated transfer of long noncoding RNA H19 induces doxorubicin resistance in breast cancer

Development of the acquired resistance is one major obstacle during chemotherapy for cancer patients. Exosomes mediate intercellular communication and cause environmental changes in tumor progression by transmitting active molecules. In this study, the role of long noncoding RNA H19 within exosomes is elucidated in terms of regulating doxorubicin (DOX) resistance of breast cancer. As a result, increased H19 expression was observed in DOX-resistant breast cancer cells in comparison with the corresponding parental cells. Suppression of H19 significantly lowered DOX resistance by decreasing cell viability, lowering colony-forming ability, and inducing apoptosis. Moreover, extracellular H19 could be moved to sensitive cells via being incorporated into exosomes. Treating sensitive cells with exosomes from resistant cells increased the chemoresistance of DOX, while downregulation of H19 in sensitive cells abated this effect. Taken together, H19 could be delivered by exosomes to sensitive cells, leading to the dissemination of DOX resistance. Our finding highlights the potential of exosomal H19 as a molecular target to reduce DOX resistance.     

肿瘤细胞来源的外泌体与恶性肿瘤的进展及化疗

外泌体是细胞分泌的一种纳米级囊泡结构,在血液、唾液、尿液等多种体液中均有分布.作为一类重要的细胞间通信分子,外泌体含有多种具有生物活性的成分,可通过多种方式在人体中发挥调节作用.目前在多种类型的细胞中均发现外泌体的存在,而肿瘤细胞来源的外泌体由于其本身的特性和功能特点,可通过微环境介导肿瘤细胞的增生、血管形成和免疫耐受,并可通过介导上皮-间质转化（epithelial-mesenchymal transition, EMT）
和胞内药物排斥反应等增加肿瘤细胞的化疗抵抗能力.同时,因其含有肿瘤细胞所分泌的特异性成分,因而可通过对外泌体中相关分子改变的检测,对疾病进行诊断和监测,并可为临床个体化用药提供新思路.     

Mammosphere-forming cells from breast cancer cell lines as a tool for the identification of CSC-like- and early progenitor-targeting drugs

Here we show that a subpopulation of MCF-7 breast cancer cells which stains pale to Toluidine Blue (Light Cells- LCs), is endowed with features of CSCs. LCs give rise to self-renewing mammospheres and express typical CSC markers; moreover this subpopulation is chemoresistant and highly tumorigenic in vivo. LCs can be identified in several other breast cancer cell lines, irrespectively of their histological origin (luminal vs mesenchymal vs basal) and represent an heterogeneous cell population composed mainly of CSC-like and early progenitor cells. By a limited in vitro drug screening assay, we identify compounds which can specifically interfere with the viability of LCs from multiple breast cancer cell lines. Analysis of the Sphere-Forming Efficiency (SFE) and of the distribution of ALDH^bright cells within the treated cell lines suggest that one of the identified compounds acts in vitro by modulating the CSC phenotype. Interestingly, a subset of the identified compounds is known to affect directly or indirectly the NFkB pathway which is emerging as an important modulator of CSC proliferation and chemoresistance.     

Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs

Adriamycin and docetaxel are two agents commonly used in treatment of breast cancer, but their efficacy is often limited by the emergence of chemoresistance. Recent studies indicate that exosomes act as vehicles for exchange of genetic cargo between heterogeneous populations of tumor cells, engendering a transmitted drug resistance for cancer development and progression. However, the specific contribution of breast cancer-derived exosomes is poorly understood. Here we reinforced other''s report that human breast cancer cell line MCF-7/S could acquire increased survival potential from its resistant variants MCF-7/Adr and MCF-7/Doc. Additionally, exosomes of the latter, A/exo and D/exo, significantly modulated the cell cycle distribution and drug-induced apoptosis with respect to S/exo. Exosomes pre-treated with RNase were unable to regulate cell cycle and apoptosis resistance, suggesting an RNA-dependent manner. Microarray and polymerase chain reaction for the miRNA expression profiles of A/exo, D/exo, and S/exo demonstrated that they loaded selective miRNA patterns. Following A/exo and D/exo transfer to recipient MCF-7/S, the same miRNAs were significantly increased in acquired cells. Target gene prediction and pathway analysis showed the involvement of miR-100, miR-222, and miR-30a in pathways implicated in cancer pathogenesis, membrane vesiculation and therapy failure. Furthermore, D/exo co-culture assays and miRNA mimics transfection experiments indicated that miR-222-rich D/exo could alter target gene expression in MCF-7/S. Our results suggest that drug-resistant breast cancer cells may spread resistance capacity to sensitive ones by releasing exosomes and that such effects could be partly attributed to the intercellular transfer of specific miRNAs.     

miR-200c-3p靶向FOSL1增加乳腺癌细胞化疗敏感性

化疗耐受是乳腺癌复发转移率居高不下、综合治疗效果难以提高的主要瓶颈。前期研究证实,miR-200c-3p在乳腺癌敏感细胞MCF-7中的表达量显著高于耐药细胞MCF-7/5Fu,提示miR-200c-3p可能参与乳腺癌化疗增敏,但是具体机制不详。生物信息学预测联合双荧光素酶报告基因实验证实,miR-200c-3p靶向调控FOSL1,且在多种肿瘤中miR-200c-3p与FOSL1表达负相关。实时荧光定量PCR技术和Western印迹技术证实,FOSL1在耐药细胞MCF-7/5Fu中的表达量显著高于亲本细胞MCF-7。在MCF-7细胞中,过表达FOSL1能够显著提高该细胞对5-Fu的化疗耐受;在MCF-7/5Fu中,使用siRNA技术沉默FOSL1,将提高该细胞对5-Fu的化疗敏感性。此外,MTT实验还发现,miR-200c-3p抑制剂能够显著上调MCF-7细胞对5-Fu的耐受,但是在此细胞中干扰FOSL1的表达,又可以增加其对5-Fu的化疗敏感性;miR-200c-3p mimics显著增加MCF-7/5Fu细胞的化疗敏感性,上调FOSL1表达后又可逆转miR-200c-3p mimics的化疗增敏作用。总之,miR-200-3p能够通过靶向FOSL1增加乳腺癌细胞对5-fluorouridine化疗敏感性。     

Chemoresistance of cancer floating cells is independent of their ability to form 3D structures: Implications for anticancer drug screening

Three-dimensional (3D) culture systems such as floating spheroids (FSs) and floating tumorspheres (FTs) are widely used as tumor models of chemoresistance. FTs are considered to be enriched in cancer stem-like cells (CS-LCs). In this study, we used cancer cell lines (lung H460, prostate LnCAP, and breast MCF-7) able to form FSs under anchorage-independent conditions and compared with cell lines (prostate PC3 and breast MDA-MB-231) that cannot form FSs under similar conditions. Independent of their ability to form FTs all cell lines growing under anchorage-independent conditions become highly resistant to obatoclax, colchicine, and hydroxyurea. We used anti-E-cadherin antibody (that blocked the formation of FSs) and demonstrated that floating LnCAP cells showed similar chemoresistance regardless of the formation of spheroids. Our results demonstrate that the development of chemoresistance is not because of the formation of a complex 3D structure and/or enrichment of CS-LCs but is likely the result of cell detachment per se and their ability to survive under anchorage-independent conditions. We propose that FSs and FTs could be useful models to study chemoresistance of cancer cells associated with cell detachment (e.g., circulating tumor cells) but they may not be representative of other types of chemoresistance that arise in vivo in attached cells.     

外泌体传递环状RNA介导肿瘤化疗耐药

环状RNA(circular RNA,circRNA)作为非编码RNA家族的重要成员,是一类共价闭环结构的单链RNA,没有多聚腺苷酸尾和5'-与-3'末端,显示出高度稳定性、丰富性和物种保守性等特点.近年来研究发现,circRNA与肿瘤化疗耐药、恶性进展等在内的多种生物学进程密切相关,发挥着极其重要的作用.外泌体是由机...     

Exosomes derived from human mesenchymal stem cells confer drug resistance in gastric cancer

Mesenchymal stem cells (MSCs) play an important role in chemoresistance. Exosomes have been reported to modify cellular phenotype and function by mediating cell-cell communication. In this study, we aimed to investigate whether exosomes derived from MSCs (MSC-exosomes) are involved in mediating the resistance to chemotherapy in gastric cancer and to explore the underlying molecular mechanism. We found that MSC-exosomes significantly induced the resistance of gastric cancer cells to 5-fluorouracil both in vivo and ex vivo. MSC-exosomes antagonized 5-fluorouracil-induced apoptosis and enhanced the expression of multi-drug resistance associated proteins, including MDR, MRP and LRP. Mechanistically, MSC-exosomes triggered the activation of calcium/calmodulin-dependent protein kinases (CaM-Ks) and Raf/MEK/ERK kinase cascade in gastric cancer cells. Blocking the CaM-Ks/Raf/MEK/ERK pathway inhibited the promoting role of MSC-exosomes in chemoresistance. Collectively, MSC-exosomes could induce drug resistance in gastric cancer cells by activating CaM-Ks/Raf/MEK/ERK pathway. Our findings suggest that MSC-exosomes have profound effects on modifying gastric cancer cells in the development of drug resistance. Targeting the interaction between MSC-exosomes and cancer cells may help improve the efficacy of chemotherapy in gastric cancer.     

Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells

Sphingosine-1-phosphate (S1P) is a potent lysolipid involved in a variety of biological responses important for cancer progression. Therefore, we investigated the role of sphingosine kinase type 1 (SphK1), the enzyme that makes S1P, in the motility, growth, and chemoresistance of MCF-7 breast cancer cells. Epidermal growth factor (EGF), an important growth factor for breast cancer progression, activated and translocated SphK1 to plasma membrane. SphK1 was required for EGF-directed motility. Downregulation of SphK1 in MCF-7 cells reduced EGF- and serum-stimulated growth and enhanced sensitivity to doxorubicin, a potent chemotherapeutic agent. These results suggest that SphK1 may be critical for growth, metastasis and chemoresistance of human breast cancers.     

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

c-MET protects breast cancer cells from apoptosis induced by sodium butyrate

Sodium butyrate (NaBu) is regarded as a potential reagent for cancer therapy. In this study, a specific breast cancer cell population that is resistant NaBu treatment was identified. These cells possess cancer stem cell characters, such as the capability of sphere formation in vitro and high tumor incident rate (85%) in mouse model. Forty percent of the NaBu resistant cells express the cancer stem cells marker, the CD133, whereas only 10% intact cells present the CD133 antigen. Furthermore, the endogenous expressing c-MET contributes to the survival of cancer stem cell population from the treatment of NaBu. The CD133+ group also presents a higher level of c-MET. A combination treatment of MET siRNA and NaBu efficiently prohibited the breast cancer progression, and the incident rate of the tumor decrease to 18%. This study may help to develop a new and alternative strategy for breast cancer therapy.     

Drug-resistant endothelial cells facilitate progression,EMT and chemoresistance in nasopharyngeal carcinoma via exosomes

Recent antitumor drug development has included investigation of a wide variety of anti-angiogenesis therapies. Because cancer cells in tumors require new blood vessels to grow and spread, they stimulate capillary proliferation from existing vessels as well as new vessel formation from endothelial precursor cells. Our previous findings suggested that drug resistance in mouse endothelial cells supported tumor growth, but the relationship between endothelial cells (ECs) and nasopharyngeal carcinoma (NPC) cells remained unclear. Exosomes are small membrane vesicles that are released by several cell types, including human microvascular ECs (HMECs). Exosomes carrying membrane and cytoplasmic constituents have been described as participants in a novel mechanism of cell-to-cell communication. In the present study, we investigated the mechanisms underlying the interactions between HMECs and NPC cells. We found that drug-resistant HMECs secreted small heterogeneous 40–100 nm vesicles, defined as exosomes. Co-incubation of NPC cells with doxorubicin-resistant (R-DOX) HMEC-derived exosomes resulted in promotion of their proliferation, migration, and chemoresistance, as well as changes in the expression of epithelial–mesenchymal transition (EMT) markers. These effects were significantly inhibited by treatment with GW4869 (an exosome inhibitor). We also found that GW4869 inhibited the stimulation of drug-resistant HMECs on NPC progression by modulating EMT in vivo. These data suggest that exosomes participate in a novel mechanism by which drug-resistant ECs enhance NPC progression.     

Fisetin induces apoptosis in breast cancer MDA‐MB‐453 cells through degradation of HER2/neu and via the PI3K/Akt pathway

Overexpression of human epidermal growth factor receptor 2 (HER2) is observed in breast cancer. The major snag faced by the human population is the development of chemoresistance to HER2 inhibitors by advanced stage breast cancer cells. Moreover, recent researchers focussed on fisetin as an antiproliferative and chemotherapeutic agent. Therefore, this study was intended to analyze the effects of fisetin on HER2/neu‐overexpressing breast cancer cell lines. Our results depicted that fisetin induced apoptosis of these cells by various mechanisms, such as inactivation of the receptor, induction of proteasomal degradation, decreasing its half‐life, decreasing enolase phosphorylation, and alteration of phosphatidylinositol 3‐kinase/Akt signaling.     

Breast cancer-derived exosomes: Tumor progression and therapeutic agents

Tumor cells secrete extracellular vesicles (EVs) for intercellular communication. EVs by transporting different proteins, nucleic acids, and lipids contribute to affect target cell function and fate. ‎EVs which originate directly from multivesicular bodies so-called exosomes have dramatically fascinated the attention of researchers owing to their ‎pivotal roles in the tumorigenesis. Breast cancer, arising from milk-producing cells, is the most identified cancer among women and has become the leading cause of cancer-related death in women globally. Although different therapies are applied to eliminate breast tumor cells, however, the efficient therapy and survival rate of patients remain challenges. Growing evidence ‎shows exosomes from breast cancer cells contribute to proliferation, metastasis, angiogenesis, chemoresistance, and also radioresistance and, thus carcinogenesis. Additionally, these exosomes may serve as a cancer treatment tool because they are a good candidate for cancer diagnosis (as biomarker) and therapy (as drug-carrier). Despite recent development in the biology of tumor-derived exosomes, the detailed mechanism of tumorigenesis, and exosome-based cancer-therapy remain still indefinable. Here, we discuss the key function of breast cancer-derived exosomes in tumorgenesis and shed light on the possible clinical application of these exosomes in breast cancer treatment.     

Potentiated DNA Damage Response in Circulating Breast Tumor Cells Confers Resistance to Chemotherapy

Circulating tumor cells (CTCs) are seeds for cancer metastasis and are predictive of poor prognosis in breast cancer patients. Whether CTCs and primary tumor cells (PTCs) respond to chemotherapy differently is not known. Here, we show that CTCs of breast cancer are more resistant to chemotherapy than PTCs because of potentiated DNA repair. Surprisingly, the chemoresistance of CTCs was recapitulated in PTCs when they were detached from the extracellular matrix. Detachment of PTCs increased the levels of reactive oxygen species and partially activated the DNA damage checkpoint, converting PTCs to a CTC-like state. Inhibition of checkpoint kinases Chk1 and Chk2 in CTCs reduces the basal checkpoint response and sensitizes CTCs to DNA damage in vitro and in mouse xenografts. Our results suggest that DNA damage checkpoint inhibitors may benefit the chemotherapy of breast cancer patients by suppressing the chemoresistance of CTCs and reducing the risk of cancer metastasis.     

Emerging role of exosome signalling in maintaining cancer stem cell dynamic equilibrium

Cancer stem cells (CSCs) are a small subset of heterogeneous cells existed in tumour tissues or cancer cell lines with self‐renewal and differentiation potentials. CSCs were considered to be responsible for the failure of conventional therapy and tumour recurrence. However, CSCs are not a static cell population, CSCs and non‐CSCs are maintained in dynamic interconversion state by their self‐differentiation and dedifferentiation. Therefore, targeting CSCs for cancer therapy is still not enough,exploring the mechanism of dynamic interconversion between CSCs and non‐CSCs and blocking the interconversion seems to be imperative. Exosomes are 30‐100 nm size in diameter extracellular vesicles (EVs) secreted by multiple living cells into the extracellular space. They contain cell‐state‐specific bioactive materials, including DNA, mRNA, ncRNA, proteins, lipids, etc. with their specific surface markers, such as, CD63, CD81, Alix, Tsg101, etc. Exosomes have been considered as information carriers in cell communication between cancer cells and non‐cancer cells, which affect gene expressions and cellular signalling pathways of recipient cells by delivering their contents. Now that exosomes acted as information carriers, whether they played role in maintaining dynamic equilibrium state between CSCs and non‐CSCs and their mechanism of activity are unknown. This review summarized the current research advance of exosomes’ role in maintaining CSC dynamic interconversion state and their possible mechanism of action, which will provide a better understanding the contribution of exosomes to dedifferentiation and stemness acquisition of non‐CSCs, and highlight that exosomes might be taken as the attractive target approaches for cancer therapeutics.     

Exosomes Released from Breast Cancer Carcinomas Stimulate Cell Movement

For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.     

Hypoxia-induced tRNA-derived fragments,novel regulatory factor for doxorubicin resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of epithelial breast malignancy, and chemoresistance is the major obstacle for cancer therapy. TNBC is associated with a hypoxic phenotype, and hypoxia contributes to the chemoresistance in breast cancer. Transfer RNA-derived fragments (tDRs) represent a new class of small noncoding RNAs that can be induced specifically by hypoxia. Here, we conducted a comparative analysis of the aberrant expression of tDRs in hypoxia-treated TNBC cell lines through the use of high-throughput sequencing technique. Quantitative real-time polymerase chain reaction was used to validate the differently expressed tDRs between two samples. The results showed that tDR-0009 [derived from transfer RNA (tRNA)^Gly-GCC-1-1] and tDR-7336 (derived from tRNA ^{Gly-GCC-1–2}) were significantly upregulated when the SUM-1315 cell lines were stimulated by hypoxia. Gene ontology (GO) and pathway analysis indicated that these two upregulated tDRs were mainly involved in maintenance of stem cell population and cellular response to interleukin (IL)-6, which may be the underlying mechanism of hypoxia-induced tDRs that facilitate the doxorubicin resistance in TNBC. The protein–protein interaction network for predicted target genes established by the STRING database manifested that tDR-0009 (tDR-7336) might be involved in the chemoresistance of TNBC via regulation of the activation of phosphorylation of STAT3. In summary, our study provided a comprehensive analysis of the deviant expression profiling of tDRs in hypoxia-treated TNBC cell lines. Specific tDRs may be a new class of regulatory factors involved in the hypoxia-induced chemoresistance in TNBC, and they could serve as potential biomarkers and intervention targets.