MEK/ERK-mediated oncogenic signals promote secretion of extracellular vesicles by controlling lysosome function.

Cancer cells secrete large amounts of extracellular vesicles (EVs) originating from multivesicular bodies (MVBs). Mature MVBs fuse either with the plasma membrane for release as EVs, often referred as to exosomes or with lysosomes for degradation. However, the mechanisms regulating MVB fate remain unknown. Here, we investigated the regulators of MVB fate by analyzing the effects of signaling inhibitors on EV secretion from cancer cells engineered to secrete luciferase-labeled EVs. Inhibition of the oncogenic MEK/ERK pathway suppressed EV release and activated lysosome formation. MEK/ERK-mediated lysosomal inactivation impaired MVB degradation, resulting in increased EV secretion from cancer cells. Moreover, MEK/ERK inhibition prevented c-MYC expression and induced the nuclear translocation of MiT/TFE transcription factors, thereby promoting the activation of lysosome-related genes, including the gene encoding a subunit of vacuolar-type H+ -ATPase, which is responsible for lysosomal acidification and function. Furthermore, c-MYC upregulation was associated with lysosomal gene downregulation in MEK/ERK-activated renal cancer cells/tissues. These findings suggest that the MEK/ERK/c-MYC pathway controls MVB fate and promotes EV production in human cancers by inactivating lysosomal function.

In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter.

Monitoring of exosome dynamics in living organisms is essential to demonstrate the real functions of cancer-derived exosomes. Currently, these have been elucidated in vitro or under non-physiological conditions in vivo in most cases. To overcome these limitations, we developed an imaging method using Antares2-mediated bioluminescence resonance energy transfer (BRET) for observing long-term accumulation of exosomes in vivo. Ectopic expression of CD63-Antares2 effectively labeled exosomes with Antares2, which emitted intense, long-wavelength luminescence suitable for in vivo monitoring. Transplantation of CD63-Antares2-expressing prostate cancer cells into mice allowed determining the amount of cancer-derived exosomes released from primary tumors into the bloodstream and visualizing the long-term homing behavior of exosomes to their target organs or tissues. Interestingly, secreted exosome was decreased upon administration of low dose of dasatinib, an approved tyrosine-kinase inhibitor. The CD63-Antares2 xenograft mouse model will be useful for elucidating the dynamics of cancer-derived exosomes in vivo and evaluating the therapeutic efficacy and mechanism of exosome production inhibitors.

Rictor promotes tumor progression of rapamycin-insensitive triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC), characterized by decreased expression of hormone receptors and human epidermal growth factor type 2 receptor, has poor prognosis and lacks effective therapeutics. Recently, the mTOR inhibitor rapamycin and its analogs have attracted growing interests and evaluated as therapeutic agents against TNBC, in which the PI3K/AKT/mTOR pathway is often activated. However, some TNBCs are less sensitive to these drugs. In this study, we found that the sensitivity of TNBC cells to rapamycin was highly dependent on the expression level of rapamycin-insensitive companion of mTOR (Rictor), a key component of the mTOR complex 2. Repression of the Rictor expression strongly suppressed the growth of rapamycin-insensitive tumor cells. Furthermore, we showed that the suppression of Rictor expression was also effective in rapamycin-insensitive cells that had acquired resistance to mTOR kinase inhibitors. These findings indicate that Rictor can be a predictive marker for the use of rapamycin analogs in TNBC and highlight the need to develop therapeutics targeting Rictor in the treatment of TNBC.

c-Src promotes tumor progression through downregulation of microRNA-129-1-3p.

MicroRNAs (miRNAs) fine-tune cellular signaling by regulating expression of signaling proteins, and aberrant expression of miRNAs is observed in many cancers. The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. In previous investigations of miRNA-mediated control of c-Src-related oncogenic pathways, we identified miRNAs that were downregulated in association with c-Src transformation and uncovered the signaling networks by predicting their target genes, which might act cooperatively to control tumor progression. Here, to further elucidate the process of cell transformation driven by c-Src, we analyzed the expression profiles of miRNAs in a doxycycline-inducible Src expression system. We found that miRNA (miR)-129-1-3p was downregulated in the early phase of c-Src-induced cell transformation, and that reexpression of miR-129-1-3p disrupted c-Src-induced cell transformation. In addition, miR-129-1-3p downregulation was tightly associated with tumor progression in human colon cancer cells/tissues. Expression of miR-129-1-3p in human colon cancer cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasion. We also identified c-Src and its critical substrate Fer, and c-Yes, a member of the Src family of kinases, as novel targets of miR-129-1-3p. Furthermore, we found that miR-129-1-3p-mediated regulation of c-Src/Fer and c-Yes is important for controlling cell adhesion and invasion. Downregulation of miR-129-1-3p by early activation of c-Src increases expression of these target genes and synergistically promotes c-Src-related oncogenic signaling. Thus, c-Src-miR-129-1-3p circuits serve as critical triggers for tumor progression in many human cancers that harbor upregulation of c-Src.

Efficient Epstein-Barr Virus Progeny Production Mediated by Cancer-Derived LMP1 and Virally-Encoded microRNAs.

Epstein-Barr virus (EBV) genomes, particularly their latent genes, are heterogeneous among strains. The heterogeneity of EBV-encoded latent membrane protein 1 (LMP1) raises the question of whether there are functional differences between LMP1 expressed by cancer-associated EBV and that by non-cancerous strains. Here, we used bacterial artificial chromosome (BAC)-cloned EBV genomes retaining all virally encoded microRNA (miRNA) genes to investigate the functions of cancer-derived LMP1 in the context of the EBV genome. HEK293 cells were stably transfected with EBV-BAC clone DNAs encoding either nasopharyngeal carcinoma (NPC)-derived CAO-LMP1 (LMP1CAO) or LMP1 from a prototype B95-8 strain of EBV (LMP1B95-8). When an EBV-BAC clone DNA encoding LMP1CAO was stably transfected into HEK293 cells, it generated many more stable transformants than the control clone encoding LMP1B95-8. Furthermore, stably transfected HEK293 cells exhibited highly efficient production of progeny virus. Importantly, deletion of the clustered viral miRNA genes compromised the ability to produce progeny viruses. These results indicate that cancer-derived LMP1 and viral miRNAs together are necessary for efficient production of progeny virus, and that the resulting increase in efficiency contributes to EBV-mediated epithelial carcinogenesis.

Src in endosomal membranes promotes exosome secretion and tumor progression.

c-Src is a membrane-associated tyrosine kinase that has key roles in the signaling transduction that controls cell growth, adhesion, and migration. In the early stage of carcinogenesis, c-Src is activated under the plasma membrane and transduces oncogenic signals. Here we show that c-Src localized to the endosomal membrane has unique functions in c-Src-transformed cells. Our results indicate that activated c-Src in the endosomal membrane promoted the secretion of exosomes, in which c-Src was encapsulated. In addition, the ESCRT-interacting molecule, Alix was identified as a c-Src-interacting protein in exosomes. We revealed that the interaction between the SH3 domain of c-Src and the proline-rich region of Alix activates ESCRT-mediated intra-luminal vesicle (ILV) formation, resulting in the upregulation of exosome secretion in c-Src-transformed cells. We observed also a correlation between malignant phenotypes and Alix-dependent aberrant exosome secretion in Src-upregulated cancer cells. Collectively, our findings provide a unique mechanism for the upregulation of exosomes in cancer cells, as well as new insights into the significance of exosome secretion in cancer progression.

Sensitive and rapid quantification of exosomes by fusing luciferase to exosome marker proteins.

Exosomes have emerged as important mediators of intercellular communication. Although their modes of action have been elucidated, the molecular mechanisms underlying their secretion, sorting of molecules, uptake into recipient cells, and biological distribution in vivo remain elusive. Here, we present a novel system for quantifying secreted exosomes by introducing ectopic or CRISPR/Cas9-mediated knock-in of luciferase-fusion exosome markers such as CD63. This luciferase-based method makes it possible to measure exosomes secreted into the culture medium with high linearity and wide dynamic range in a high-throughput manner. We demonstrate that data obtained by luminescent quantification are well correlated with data obtained by conventional nanoparticle tracking analysis under multiple conditions. In addition, our system is capable of evaluating the recipient cells or tissues that take up exosomes, as well as visualizing exosomes in vivo. The proposed system represents a powerful tool for understanding the molecular mechanisms underlying exosome production, uptake, and long-term distribution.

Clustered microRNAs of the Epstein-Barr virus cooperatively downregulate an epithelial cell-specific metastasis suppressor.

UNLABELLED: The Epstein-Barr virus (EBV) encodes its own microRNAs (miRNAs); however, their biological roles remain elusive. The commonly used EBV B95-8 strain lacks a 12-kb genomic region, known as BamHI A rightward transcripts (BART) locus, where a number of BART miRNAs are encoded. Here, bacterial artificial chromosome (BAC) technology was used to generate an EBV B95-8 strain in which the 12-kb region was fully restored at its native locus [BART(+) virus]. Epithelial cells were stably infected with either the parental B95-8 virus or the BART(+) virus, and BART miRNA expression was successfully reconstituted in the BART(+) virus-infected cells. Microarray analyses of cellular gene expression identified N-myc downstream regulated gene 1 (NDRG1) as a putative target of BART miRNAs. The NDRG1 protein was barely expressed in B cells, highly expressed in epithelial cells, including primary epithelial cells, and strongly downregulated in the BART(+) virus-infected epithelial cells of various origins. Although in vitro reporter assays identified BART22 as being responsible for the NDRG1 downregulation, EBV genetic analyses revealed that BART22 was not solely responsible; rather, the entire BART miRNA cluster 2 was responsible for the downregulation. Immunohistochemical analyses revealed that the expression level of the NDRG1 protein was downregulated significantly in EBV-positive nasopharyngeal carcinoma specimens. Considering that NDRG1 encodes an epithelial differentiation marker and a suppressor of metastasis, these data implicate a causative relationship between BART miRNA expression and epithelial carcinogenesis in vivo. IMPORTANCE: EBV-related epithelial cancers, such as nasopharyngeal carcinomas and EBV-positive gastric cancers, encompass more than 80% of EBV-related malignancies. Although it is known that they express high levels of virally encoded BART miRNAs, how these miRNAs contribute to EBV-mediated epithelial carcinogenesis remains unknown. Although a number of screenings have been performed to identify targets of viral miRNAs, many targets likely have not been identified, especially in case of epithelial cell infection. This is the first study to use EBV genetics to perform unbiased screens of cellular genes that are differentially expressed in viral miRNA-positive and -negative epithelial cells. The result indicates that multiple EBV-encoded miRNAs cooperatively downregulate NDRG1, an epithelial differentiation marker and suppressor of metastasis. The experimental system described in this study should be useful for further clarifying the mechanism of EBV-mediated epithelial carcinogenesis.