Recipient Cancer Cells Research Articles

High-metastatic cancer cells derived exosomal miR92a-3p promotes epithelial-mesenchymal transition and metastasis of low-metastatic cancer cells by regulating PTEN/Akt pathway in hepatocellular carcinoma

Exosomes play an important role in intercellular communication and metastatic progression of hepatocellular carcinoma (HCC). However, cellular communication between heterogeneous HCC cells with different metastatic potentials and the resultant cancer progression are not fully understood in HCC. Here, HCC cells with high-metastatic capacity (97hm and Huhm) were constructed by continually exerting selective pressure on primary HCC cells (MHCC-97H and Huh7). Through performing exosomal miRNA sequencing in HCC cells with different metastatic potentials (MHCC-97H and 97hm), many significantly different miRNA candidates were found. Among these miRNAs, miR-92a-3p was the most abundant miRNA in the exosomes of highly metastatic HCC cells. Exosomal miR92a-3p was also found enriched in the plasma of HCC patient-derived xenograft mice (PDX) model with high-metastatic potential. Exosomal miR-92a-3p promotes epithelial-mesenchymal transition (EMT) in recipient cancer cells via targeting PTEN and regulating its downstream Akt/Snail signaling. Furthermore, through mRNA sequencing in HCC cells with different metastatic potentials and predicting potential transcription factors of miR92a-3p, upregulated transcript factors E2F1 and c-Myc were found in high-metastatic HCC cells promote the expression of cellular and exosomal miR-92a-3p in HCC by directly binding the promoter of its host gene, miR17HG. Clinical data showed that a high plasma exosomal miR92a-3p level was correlated with shortened overall survival and disease-free survival, indicating poor prognosis in HCC patients. In conclusion, hepatoma-derived exosomal miR92a-3p plays a critical role in the EMT progression and promoting metastasis by inhibiting PTEN and activating Akt/Snail signaling. Exosomal miR92a-3p is a potential predictive biomarker for HCC metastasis, and this may provoke the development of novel therapeutic and preventing strategies against metastasis of HCC.

Senescent Stromal Cells Promote Cancer Resistance through SIRT1 Loss-Potentiated Overproduction of Small Extracellular Vesicles.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype, which is implicated in age-related pathologies including cancer. Here, we report that senescent cells actively synthesize and release small extracellular vesicles (sEV) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP-binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathologic activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by cotargeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. SIGNIFICANCE: Senescent stromal cells produce a large number of sEVs to promote cancer resistance in therapeutic settings, a process driven by SIRT1 decline in stromal cells and ABCB4 augmentation in cancer cells.See related commentary by Wiley, p. 3193 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/16/3383/F1.large.jpg.

Abstract 2969: Senescent stromal cells promote cancer resistance via Sirt1 loss-potentiated biogenesis of small extracellular vesicles

Abstract Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype (SASP) and implicated in age-related pathologies including cancer. Here we report that senescent cells actively synthesize and release small extracellular vesicles (sEVs) with a distinctive size distribution. Mechanistically, SIRT1 loss supports accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Senescent stromal cell-derived sEVs carry a special set of cargoes, including small RNA species such as microRNAs (miRNAs) whose spectrum markedly differ from that of proliferating cell-secreted sEVs. Once released, senescent stromal sEVs significantly alter the expression profile of recipient cancer cells and enhance their aggressiveness, specifically drug resistance mediated by expression of ATP binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with an agonist SRT2104 prevents development of cancer resistance through restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients are readily detectable by routine biotechniques, presenting a novel biomarker to monitor therapeutic efficacy and predict long term outcome. Together, our study identifies a distinct mechanism supporting pathological activities of senescent cells, and provides a novel avenue to circumvent advanced human malignancies by co-targeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. Citation Format: Liu Han, Qilai Long, Eric Lam, Yu Sun. Senescent stromal cells promote cancer resistance via Sirt1 loss-potentiated biogenesis of small extracellular vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2969.

Epithelial cell -derived microvesicles: A safe delivery platform of CRISPR/Cas9 conferring synergistic anti-tumor effect with sorafenib

Safe and efficient intracellular delivery of CRISPR/Cas9 is a key step for effective therapeutic genome editing in a wide range of diseases. This remains challenging due to multiple drawbacks of the currently available vehicles. Here we report that epithelial cell -derived microvesicles (MVs) function as safe and natural carriers for efficient delivery of CRISPR/Cas9 to treat cancer. In our study, compared to epithelial cell -derived MVs, cancer -derived MVs were quickly absorbed intracellularly by recipient cancer cells in vitro and showed selective accumulation in tumors of HepG2 xenografts in vivo, due to their cancer cell tropism dependent targeting. Surprisingly, synergistic anti-tumor effect of sgIQ 1.1 loaded Cas9MVs/HEK293 + sorafenib was better than sgIQ 1.1 + Cas9MVs/HepG2 + sorafenib in vitro. In addition, qPCR results showed that miR-21 and miR-181a expression were upregulated in HepG2 cells treated with cancer cell -derived MVs that might support the cancer progression. Further, treatment of HepG2 xenografts with sgIQ 1.1 loaded Cas9MVs/HEK293 showed enhanced anti-cancer effect than sgIQ 1.1 + Cas9MVs/HepG2. Therefore, we conclude that normal cells -derived MVs can act as better and safe natural delivery systems for cancer therapeutics in the future.

Exosomes carrying ALDOA and ALDH3A1 from irradiated lung cancer cells enhance migration and invasion of recipients by accelerating glycolysis.

Lung cancer has been recognized as the leading cause of cancer-related death worldwide. Despite the improvements of treatment, the distant metastasis and recurrence of lung cancer caused by therapy resistance is the biggest challenge in clinical management. Extracellular vesicles named exosomes play crucial roles in intercellular communication as signaling mediators and are involved in tumor development. In this study, we isolated exosomes from irradiated lung cancer cells and co-cultured the exosomes with other lung cancer cells. It was found that cellular growth and motility of recipient cells were facilitated. High-throughput LC-MS/MS assay of exosomal proteins and Gene Ontology enrichment analyses indicated that the metabolic enzymes ALDOA and ALDH3A1 had potential contribution in exosome-enhanced motility of recipient cells, and clinical survival analysis demonstrated the close correlations between ALDOA or ALDH3A1 expression and poor prognosis of lung cancer patients. After co-culturing with exosomes derived from irradiated cancer cells, the expressions of these metabolic enzymes were elevated and the glycolytic activity was promoted in recipient cancer cells. In conclusion, our data suggested that exosomes from irradiated lung cancer cells regulated the motility of recipient cells by accelerating glycolytic process, where exosomal ALDOA and ALDH3A1 proteins were important signaling factors.

Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells

ABSTRACT The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVβ3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from αVβ3-expressing cells affect tumour growth differently than sEVs from control cells that do not express αVβ3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVβ3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVβ3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.

Targeting amphiregulin (AREG) derived from senescent stromal cells diminishes cancer resistance and averts programmed cell death 1 ligand (PD-L1)-mediated immunosuppression.

Aging is characterized by a progressive loss of physiological integrity, while cancer represents one of the primary pathological factors that severely threaten human lifespan and healthspan. In clinical oncology, drug resistance limits the efficacy of most anticancer treatments, and identification of major mechanisms remains a key to solve this challenging issue. Here, we highlight the multifaceted senescence‐associated secretory phenotype (SASP), which comprises numerous soluble factors including amphiregulin (AREG). Production of AREG is triggered by DNA damage to stromal cells, which passively enter senescence in the tumor microenvironment (TME), a process that remarkably enhances cancer malignancy including acquired resistance mediated by EGFR. Furthermore, paracrine AREG induces programmed cell death 1 ligand (PD‐L1) expression in recipient cancer cells and creates an immunosuppressive TME via immune checkpoint activation against cytotoxic lymphocytes. Targeting AREG not only minimized chemoresistance of cancer cells, but also restored immunocompetency when combined with classical chemotherapy in humanized animals. Our study underscores the potential of in vivo SASP in driving the TME‐mediated drug resistance and shaping an immunosuppressive niche, and provides the proof of principle of targeting major SASP factors to improve therapeutic outcome in cancer medicine, the success of which can substantially reduce aging‐related morbidity and mortality.

Abstract 3819: Amphiregulin in damaged tumor microenvironment potentiates therapeutic resistance and enables PD-L1-mediated immunosuppression

Abstract Therapeutic resistance is the major barrier to conquer human cancer, the most lethal pathology that claims numerous lives per year. Although identification of resistance mechanisms is the key to develop optimal strategies, progress has been limited due to long term failure to reveal the most critical targets. Cancer cells exhibit high plasticity even in clinical settings, a phenomenon that contributes to disease refraction by developing intrinsic or acquired resistance. Here we highlight the multifaceted and genetically conserved senescence-associated secretory phenotype (SASP), which encodes a large array of soluble factors including amphiregulin (AREG). Expression of AREG is markedly inducible by DNA damage to stromal cells in the tumor microenvironment (TME), a process mediated predominantly by the NF-κB complex and C/EBP family. Stromal AREG remarkably enhances the malignancy of cancer cells including acquired resistance, mainly by activating the EGFR pathway and causing a distinct epithelial to endothelial transition (EET). Paracrine AREG induces PD-L1 expression in recipient cancer cells, thereby creating an immunosuppressive TME via immune checkpoint activation. Therapeutic targeting AREG by a monoclonal antibody not only remarkably deprived cancer cells of chemoresistance, but achieved prominent efficiency when combined with classical chemotherapy in humanized animals with restored immunocompetency. Stromal AREG enters circulation and is readily detectable in the peripheral blood of post-treatment patients, representing a novel liquid biopsy biomarker for therapeutic evaluation and disease prognosis. Our study substantiates the potential of in vivo SASP in driving the TME-mediated drug resistance and shaping an immunosuppressive niche, and provides the proof-of-principle of targeting major SASP factors to effectively improve therapeutic index with reduced mortality. Citation Format: Qixia Xu, Jean-Philippe Coppé, Judith Campisi, Eric Lam, Yu Sun. Amphiregulin in damaged tumor microenvironment potentiates therapeutic resistance and enables PD-L1-mediated immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3819.

Snail1-dependent cancer-associated fibroblasts induce epithelial-mesenchymal transition in lung cancer cells via exosomes.

Epithelial-mesenchymal transition (EMT) is an essential component of metastasis. Our previous study demonstrated that cancer-associated fibroblasts (CAFs) induce EMT in lung cancer cells. In recent years, many studies have demonstrated that CAFs induce metastasis and drug resistance in cancer cells via exosomes. We sought to discover the mechanism underlying how CAFs induce EMT in lung cancer cells, unveiling the role of exosomes in lung cancer progression. We cultured lung cancer cell (i) with control medium, normal fibroblasts (NFs) or CAFs; (ii) with SNAI1-transfected or NC (negative control)-transfected CAFs; (iii) with exosomes extracted from NF- or CAF-conditioned medium; (iv) with exosomes released by SNAI1 or NC-transfected CAFs; (v) with CAF-conditioned medium or exosome-depleted CAF-conditioned medium. qRT-PCR was conducted to examine the expression of CDH1 (gene of E-cadherin) and VIM (gene of Vimentin), western blotting was conducted to examine E-cadherin and vimentin levels in lung cancer cells. Exosomes released by CAFs-promoted EMT in lung cancer cells. Interestingly, SNAI1 levels in exosomes secreted from CAFs were correlated with SNAI1 expression in CAFs. Furthermore, the level of SNAI1 in exosomes was crucial for inducing EMT in lung cancer cells. Finally, treatment of CAFs with GW4869, an inhibitor of exosome release, noticeably inhibited their EMT-inducing effect on recipient epithelial cells. The molecular mechanism underlying how CAFs induce EMT in cancer cells may be that CAFs deliver SNAI1 to recipient cancer cells via exosomes.

Programming of macrophages by UV-irradiated apoptotic cancer cells inhibits cancer progression and lung metastasis

Apoptotic cell clearance by phagocytes is essential in tissue homeostasis. We demonstrated that conditioned medium (CM) from macrophages exposed to apoptotic cancer cells inhibits the TGFβ1-induced epithelial–mesenchymal transition (EMT), migration, and invasion of cancer cells. Apoptotic 344SQ (ApoSQ) cell-induced PPARγ activity in macrophages increased the levels of PTEN, which was secreted in exosomes. Exosomal PTEN was taken up by recipient lung cancer cells. ApoSQ-exposed CM from PTEN knockdown cells failed to enhance PTEN in 344SQ cells, restore cellular polarity, or exert anti-EMT and anti-invasive effects. The CM that was deficient in PPARγ ligands, including 15-HETE, lipoxin A4, and 15d-PGJ2, could not reverse the suppression of PPARγ activity or the PTEN increase in 344SQ cells and consequently failed to prevent the EMT process. Moreover, a single injection of ApoSQ cells inhibited lung metastasis in syngeneic immunocompetent mice with enhanced PPARγ/PTEN signaling both in tumor-associated macrophages and in tumor cells. PPARγ antagonist GW9662 reversed the signaling by PPARγ/PTEN; the reduction in EMT-activating transcription factors, such as Snai1 and Zeb1; and the antimetastatic effect of the ApoSQ injection. Thus, the injection of apoptotic lung cancer cells may offer a new strategy for the prevention of lung metastasis.

The exosomal compartment protects epidermal growth factor receptor from small molecule inhibitors

Epidermal growth factor receptor (EGFR) plays a significant role in promoting breast cancer progression. However, targeting EGFR as a single treatment only resulted in moderate efficacy to the disease. The underlying mechanism of low responsiveness to EGFR inhibition remains largely unclear. Tumor-secreted extracellular vesicles (EVs) play a crucial role in mediating intercellular communication between tumor and stromal cells in local microenvironment and distant metastatic niche. Extracellular vesicles mediate cell-to-cell transfer of lipids, nucleic acids, and proteins. Although numerous recent studies have demonstrated exchanges of extracellular vesicles between cancer cells and the recipient cells contribute to tumor proliferation, invasion, and metastasis, yet little is known how the exosomal compartment responds to targeted therapies and their role in promoting drug resistance. In the current study we used a triple-negative breast cancer model to show that EV-encapsulated EGFR is protected from targeted inhibitors of EGFR and can trigger signaling pathway in recipient cancer cells, promoting proliferation and migration ability in vitro. Taken together, our study suggested a novel mechanism of drug resistance entailing the EV compartment, such as exosomes, as a target shelter which when released can signal for tumor promotion in the recipient cancer cells.

Exosomes impact survival to radiation exposure in cell line models of nervous system cancer.

Radiation is utilized in the therapy of more than 50% of cancer patients. Unfortunately, many malignancies become resistant to radiation over time. We investigated the hypothesis that one method of a cancer cell's ability to survive radiation occurs through cellular communication via exosomes. Exosomes are cell-derived vesicles containing DNA, RNA, and protein. Three properties were analyzed: 1) exosome function, 2) exosome profile and 3) exosome uptake/blockade. To analyze exosome function, we show radiation-derived exosomes increased proliferation and enabled recipient cancer cells to survive radiation in vitro. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in an in vivo model. To address the mechanism underlying the alterations by exosomes in recipient cells, we obtained a profile of radiation-derived exosomes that showed expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes contain elevated oncogenic miR-889, oncogenic mRNAs, and proteins of the proteasome pathway, Notch, Jak-STAT, and cell cycle pathways. Radiation-derived exosomes contain decreased levels of tumor-suppressive miR-516, miR-365, and multiple tumor-suppressive mRNAs. Ingenuity pathway analysis revealed the most represented networks included cell cycle, growth/survival. Upregulation of DNM2 correlated with increased exosome uptake. To analyze the property of exosome blockade, heparin and simvastatin were used to inhibit uptake of exosomes in recipient cells resulting in inhibited induction of proliferation and cellular survival. Because these agents have shown some success as cancer therapies, our data suggest their mechanism of action could be limiting exosome communication between cells. The results of our study identify a novel exosome-based mechanism that may underlie a cancer cell's ability to survive radiation.

Tumor-associated macrophages-derived exosomes promote the migration of gastric cancer cells by transfer of functional Apolipoprotein E

Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment and have been shown to contribute to tumor aggressiveness. However, the detailed mechanisms underlying the pro-metastatic effect of TAMs on gastric cancer are not clearly defined. Here, we show that TAMs are enriched in gastric cancer. TAMs are characterized by M2-polarized phenotype and promote migration of gastric cancer cells in vitro and in vivo. Furthermore, we find that M2-derived exosomes determine the TAMs-mediated pro-migratory activity. Using mass spectrometry, we identify that apolipoprotein E (ApoE) is highly specific and effective protein in M2 macrophages-derived exosomes. Moreover, TAMs are uniquely immune cells population expressed ApoE in gastric cancer microenvironment. However, exosomes derived from M2 macrophages of Apoe−/− mice have no significant effect on the migration of gastric cancer cells in vitro and in vivo. Mechanistically, M2 macrophage-derived exosomes mediate an intercellular transfer of ApoE-activating PI3K-Akt signaling pathway in recipient gastric cancer cells to remodel the cytoskeleton-supporting migration. Collectively, our findings signify that the exosome-mediated transfer of functional ApoE protein from TAMs to the tumor cells promotes the migration of gastric cancer cells.

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8

ABSTRACTCancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.

Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells

Radiation is a highly efficient therapy in squamous head and neck carcinoma (HNSCC) treatment. However, local recurrence and metastasis are common complications. Recent evidence shows that cancer-cell-derived exosomes modify tumour cell movement and metastasis. In this study, we link radiation-induced changes of exosomes to their ability to promote migration of recipient HNSCC cells. We demonstrate that exosomes isolated from irradiated donor cells boost the motility of the HNSCC cells BHY and FaDu. Molecular data identified enhanced AKT-signalling, manifested through increased phospho-mTOR, phospho-rpS6 and MMP2/9 protease activity, as underlying mechanism. AKT-inhibition blocked the pro-migratory action, suggesting AKT-signalling as key player in exosome-mediated migration. Proteomic analysis of exosomes isolated from irradiated and non-irradiated BHY donor cells identified 39 up- and 36 downregulated proteins. In line with the observed pro-migratory effect of exosomes isolated from irradiated cells protein function analysis assigned the deregulated exosomal proteins to cell motility and AKT-signalling. Together, our findings demonstrate that exosomes derived from irradiated HNSCC cells confer a migratory phenotype to recipient cancer cells. This is possibly due to radiation-regulated exosomal proteins that increase AKT-signalling. We conclude that exosomes may act as driver of HNSCC progression during radiotherapy and are therefore attractive targets to improve radiation therapy strategies.

Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells.

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

Vesiculated Long Non-Coding RNAs: Offshore Packages Deciphering Trans-Regulation between Cells, Cancer Progression and Resistance to Therapies.

Extracellular vesicles (EVs) are nanosized vesicles secreted from virtually all cell types and are thought to transport proteins, lipids and nucleic acids including non-coding RNAs (ncRNAs) between cells. Since, ncRNAs are central to transcriptional regulation during developmental processes; eukaryotes might have evolved novel means of post-transcriptional regulation by trans-locating ncRNAs between cells. EV-mediated transportation of regulatory elements provides a novel source of trans-regulation between cells. In the last decade, studies were mainly focused on microRNAs; however, functions of long ncRNA (lncRNA) have been much less studied. Here, we review the regulatory roles of EV-linked ncRNAs, placing a particular focus on lncRNAs, how they can foster dictated patterns of trans-regulation in recipient cells. This refers to envisaging novel mechanisms of epigenetic regulation, cellular reprogramming and genomic instability elicited in recipient cells, ultimately permitting the generation of cancer initiating cell phenotypes, senescence and resistance to chemotherapies. Conversely, such trans-regulation may introduce RNA interference in recipient cancer cells causing the suppression of oncogenes and anti-apoptotic proteins; thus favoring tumor inhibition. Collectively, understanding these mechanisms could be of great value to EV-based RNA therapeutics achieved through gene manipulation within cancer cells, whereas the ncRNA content of EVs from cancer patients could serve as non-invasive source of diagnostic biomarkers and prognostic indicators in response to therapies.

Abstract 947: Polycomb protein SUZ12 is regulated by a novel miRNA like RNA (miR-G655A) present in extracellular vesicles secreted by mesenchymal stem/tromal cells

Abstract Introduction: Role of human mesenchymal stem/stromal cells (hMSCs) in cancer progression is well studied. Previously, we showed that hMSCs use autophagy to survive in adverse environment (e.g. serum deprivation). Using this model to mimic the solid tumor core, we1ve shown that the secretome of these stressed hMSCs is also tumor supportive. Furthermore, we showed that extracellular vesicles (EVs) secreted by cells contain small RNAs, proteins and lipids that are tumor supportive. In this study, we focused on a new and unknown small RNA contained in EVs, its potential mRNA targets in recipient cancer cells and its regulation in parent hMSCs and other cancer cells. Methods: EVs were isolated from serum deprived human mesenchymal stem/stromal cells (hMSCs) using a concentrator followed by serial ultracentrifugation. RNA molecules less that 200 bases were isolated and a purified cDNA library was used for cluster generation then sequenced on Illumina GAIIx. Human cancer cells (osteosarcoma, fibrosarcoma and breast cancer) miRNA knockdown models were obtained using agomirs and shRNA strategies against miRNA-G655A. Cell proliferation assays were performed using trypan blue and Cyquant DNA detection. In vivo tumor progression in nude mice was tracked by caliper measurement and relative GFP signal detected with in vivo imaging station. Small RNA targets were revealed by Affimetrix Human gene 2.0 ST array analysis and confirmed by 31UTR luciferase assays. Cross linked-ChIP assays for EZH2 and SUZ12 were used to identify interactions with the new small RNA promoter region. Results: Next generation sequencing of EVs from hMSCs identified an unknown small RNA, temporarily named miR-G665A. We report here that miR-G665A is conserved in mammals and its expression is confirmed, but varies between tissues or cell line1s. EVs secreted by hMSCs are notably enriched with miR-G665A. Both in vitro and in vivo knockdown studies using agomirs and shRNA strategies indicate that miR-G665A participate in cell proliferation and tumor progression. Its expression is also regulated through cell cycle. Microarray data from these knockdown studies revealed potential end targets of miR-G665A (e.g. interleukins,MMPs). Some direct targets are validated using 31UTR luciferase assays. One of the direct target is SUZ12, a component of the polycomb group complex PRC2, a master epigenetic regulator which contributes to the neoplastic phenotype. We also show that PRC2 complex (SUZ12 and EZH2) targets the regulation of the expression of this novel small RNA in a feedback loop mechanism. Conclusion: The role of this novel small RNA on the regulation of an essential transcription factor could lead to a better understanding of the crosstalk mechanisms between mesenchymal stem/stromal and cancer cells. This work is supported by NIH grant CA1515851 and the UMMC Cancer Institute startup funds. Citation Format: Patrice Penfornis, Joseph D. Fernandes, Radhika R. Pochampally. Polycomb protein SUZ12 is regulated by a novel miRNA like RNA (miR-G655A) present in extracellular vesicles secreted by mesenchymal stem/tromal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 947.

Abstract 5089: Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis

Abstract The tumor microenvironment is a crucial player in tumorigenesis, proliferation, and survival. Communication between its various components through microvesicular trafficking is central to the interaction between tumor cells and the surrounding milieu. Exosomes, nanovesicles released by all cell types, carry various proteins and nucleic acids. Once released into the extracellular environment, exosomes are taken up by surrounding cells or the exosomes travel in various bodily fluids to reach distant parts of the body. Those exosomes derived from tumor cells contain proteins and nucleic acids essential to the development and maintenance of tumors. One such protein, Survivin, a member of the Inhibitor of Apoptosis (IAP) protein family, has been identified in virtually all cancer cells. Survivin function is closely linked with subcellular location. Nuclear Survivin plays a key role in cell division and cell cycle regulation as a member of the chromosome passenger complex. Cytosolic and mitochondrial Survivin primarily facilitate the inhibition of apoptosis. Recent research has identified a unique pool of Survivin in the extracellular environment. Functions tied to this location are as yet undefined, however, evidence suggests that this population may contribute to increased proliferation, invasiveness and therapeutic resistance when taken into recipient cancer cells. Using tandem affinity purification we have identified a relationship between Survivin and TNF receptor 1 (TNFR1), as well as transferrin receptor 2 (TfR2). Both of these receptors play a role in endocytosis of extracellular proteins. Transferrin receptors are upregulated on cancer cells to facilitate the increased iron demand of proliferating cells. We therefore hypothesize that exosomal Survivin is taken up by adjacent cancer cells via receptor-mediated endocytosis. We have shown that in the presence of antibodies to these receptors, there is a decreased uptake of extracellular Survivin (from conditioned media). Results were similar when conditioned media was introduced to HeLa cells with siRNA knockdowns of each receptor, as well as to CHO cells deficient in these receptors. We conclude that uptake of extracellular Survivin into cancer cells is dependent upon these two endocytosis receptors. The relationship of Survivin and exosomes in the extracellular environment is indicative that exosomes transport Survivin out of tumor cells and into the surroundings. These results suggest that exosomal Survivin enters cancer cells by way of receptor-mediated endocytosis. Understanding this mechanism of signaling between cancer cells shows the potential intercellular trafficking of oncoproteins may play in tumor maintenance and progression. It also provides us with a novel means to combat the resulting enhanced aggressive nature of the disease. Citation Format: Amber Gonda, Salma Khan, Heather R. Ferguson Bennit, Ron B. Moyron, Nathan R. Wall. Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5089.

Exosomes from the tumor microenvironment as reciprocal regulators that enhance prostate cancer progression

Distant organ metastasis of prostate cancer is a puzzle, and various theories have successively arisen to explain the mechanism of lethal cancer progression. While perhaps agreeable to many cancer biologists, the very statement of "seed and soil" proposed by Stephan Paget in 1881 is arguably still the major statement for organ-specific cancer metastasis. Since recent studies showed important correlations of regulation of cancer cells and the microenvironment, exosomes from cancer and stromal cells seem to create another important niche for metastasis. Stromal cells pretreated with exosomes from metastatic cancer cells increase the potential of change stromal cells. The poorly metastatic cancer cells could also enhance malignancy through transfer of proteins, microribonucleic acid and messenger ribonucleic acid to recipient cancer cells. Herein, we reviewed extracellular exosomes as a factor involved in cross-talk between stromal and prostate cancer epithelial cells.