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.

Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific αvβ6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of αvβ6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1-HMEC1); we demonstrate that de novo αvβ6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the αvβ6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from β6 negative PC3 cells, generated by shRNA against β6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of β6, causes up-regulation of pSTAT1. Overall, our findings suggest that αvβ6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVβ3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVβ3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVβ3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVβ6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVβ3 in their NEPrCa primary tumors. In contrast, the αVβ6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVβ3, but not αVβ6, and the neuronal marker synaptophysin; it also demonstrates that αVβ3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVβ3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVβ6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVβ3 integrin, but not αVβ6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.

a b s t r a c tThe structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl-tRNA and mRNA movement and with the Pi release occurring after ribosome-and EF-G-dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit ''head'' which accompanies the ribosome-unlocking step of translocation.

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we demonstrate that IF3 is not involved in any of the recycling reactions but simply prevents the re-association of split ribosomal subunits. Our finding demonstrates that the important function of RRF includes the release of mRNA, which is often missed by the use of a short ORF with the Shine-Dalgarno sequence near the termination site.

Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins. It has recently been established that standard isolation techniques, such as differential ultracentrifugation, yield a mixed population of EVs. However, density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris. Here, we describe the most common methods used to isolate sEVs from cell culture medium, mouse and human plasma, and a new technique for isolating sEVs from tissues as well. This article also provides detailed procedures to isolate LEVs.

The αVβ6 integrin, an epithelial-specific cell surface receptor absent in normal prostate and expressed during prostate cancer (PrCa) progression, is a therapeutic target in many cancers. Here, we report that transcript levels of ITGB6 (encoding the β6 integrin subunit) are significantly increased in metastatic castrate-resistant androgen receptor-negative prostate tumors compared to androgen receptor-positive prostate tumors. In addition, the αVβ6 integrin protein levels are significantly elevated in androgen receptor-negative PrCa patient derived xenografts (PDXs) compared to androgen receptor-positive PDXs. In vitro , the androgen receptor-negative PrCa cells express high levels of the αVβ6 integrin compared to androgen receptor-positive PrCa cells. Additionally, expression of androgen receptor (wild type or variant 7) in androgen receptor-negative PrCa cells downregulates the expression of the β6 but not αV subunit compared to control cells. We demonstrate an efficient strategy to therapeutically target the αVβ6 integrin during PrCa progression by using short interfering RNA (siRNA) loaded into PrCa cell-derived small extracellular vesicles (sEVs). We first demonstrate that fluorescently-labeled siRNAs can be efficiently loaded into PrCa cell-derived sEVs by electroporation. By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into PrCa cells. We show that sEV-mediated delivery of ITGB6 -targeting siRNAs into PC3 cells specifically downregulates expression of the β6 subunit. Furthermore, treatment with sEVs encapsulating ITGB6 siRNA significantly reduces cell adhesion and migration of PrCa cells on an αVβ6-specific substrate, LAP-TGFβ1. Our results demonstrate an approach for specific targeting of the αVβ6 integrin in PrCa cells using sEVs encapsulating ITGB6 -specific siRNAs.

Androgen deprivation therapies aimed to target prostate cancer (PrCa) are only partially successful given the occurrence of neuroendocrine PrCa (NEPrCa), a highly aggressive and highly metastatic form of PrCa, for which there is no effective therapeutic approach. Our group has demonstrated that while absent in prostate adenocarcinoma, the αVβ3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVβ3 that promotes NE differentiation (NED). This novel pathway requires the expression of a GPI-linked surface molecule, NgR2, also known as Nogo-66 receptor homolog 1. We show here that NgR2 is upregulated by αVβ3, to which it associates; we also show that it promotes NED and anchorage-independent growth, as well as a motile phenotype of PrCa cells. Given our observations that high levels of αVβ3 and, as shown here, of NgR2 are detected in human and mouse NEPrCa, our findings appear to be highly relevant to this aggressive and metastatic subtype of PrCa. This study is novel because NgR2 role has only minimally been investigated in cancer and has instead predominantly been analyzed in neurons. These data thus pave new avenues toward a comprehensive mechanistic understanding of integrin-directed signaling during PrCa progression toward a NE phenotype.