The different prostate cancer (PCa) cell populations (bulk and cancer stem cells, CSCs) release exosomes that contain miRNAs that could modify the local or premetastatic niche. The analysis of the differential expression of miRNAs in exosomes allows evaluating the differential biological effect of both populations on the niche, and the identification of potential biomarkers and therapeutic targets. Five PCa primary cell cultures were established to originate bulk and CSCs cultures. From them, exosomes were purified by precipitation for miRNAs extraction to perform a comparative profile of miRNAs by next generation sequencing in an Illumina platform. 1839 miRNAs were identified in the exosomes. Of these 990 were known miRNAs, from which only 19 were significantly differentially expressed: 6 were overexpressed in CSCs and 13 in bulk cells exosomes. miR-100-5p and miR-21-5p were the most abundant miRNAs. Bioinformatics analysis indicated that differentially expressed miRNAs are highly related with PCa carcinogenesis, fibroblast proliferation, differentiation and migration, and angiogenesis. Besides, miRNAs from bulk cells affects osteoblast differentiation. Later, their effect was evaluated in normal prostate fibroblasts (WPMY-1) where transfection with miR-100-5p, miR-21-5p and miR-139-5p increased the expression of metalloproteinases (MMPs) -2, -9 and -13 and RANKL and fibroblast migration. The higher effect was achieved with miR21 transfection. As conclusion, miRNAs have a differential pattern between PCa bulk and CSCs exosomes that act collaboratively in PCa progression and metastasis. The most abundant miRNAs in PCa exosomes are interesting potential biomarkers and therapeutic targets.
Introduction: Exosomes from bulk and cancer stem cells (CSCs) from prostate cancer primary cultures present a differential pattern of miRNAs. The most abundant miRNA in all exosomes is miR-100, while miR-21 and miR-139 were the most abundant differentially expressed miRNAs in bulk and CSCs exosomes, respectively. Bioinformatics analysis suggested that these miRNAs were related with tumor progression. The functional effect of these miRNAs on targets related with changes at primary and pre-metastatic microenvironment was evaluated in prostatic stromal cells and osteoblasts. Methods: Normal human undifferentiated osteoblasts (hFOB1.19) and prostate fibroblasts (WPMY-1) cell lines were transfected, by separated, with 25 nM of the following miRNas: miR-100-5p, miR-21-5p, miR-139-5p. let7c was included as transfection control. After 48 hours, changes in the expression of metalloproteinases (MMP)-2, -9 and-13, RANKL, OPG and IL-8 at mRNAs and protein levels were evaluated by qPCR and western blot, respectively. As transfection control, expression of specific targets previously described for each miRNA was evaluated. Besides, the effect of miRNAs in fibroblast migration was evaluated by transwell assay. Results: In fibroblasts, transfection with miR-21, -100 and -139 increased significantly expression of RANKL, IL8 and all MMPs, with a higher effect in MMP-9. Besides, miR-21 increased fibroblast migration. In osteoblasts, transfection with miRNAs changed the balance that triggers vicious circle at the bone, increasing RANKL and diminishing OPG expression. miRNAs also increased MMPs expression. The greater effect was observed with miR-21 transfection. Conclusions: Through exosomes, prostate cancer cells recruit normal cells to favor their growth and spread. miRNAs in exosomes increase expression of proteins (in particular, MMPs and RANKL) and cell migration of fibroblasts that create a favorable microenvironment for tumor progression at the primary niche. Besides, through bloodstream, tumoral exosomes can reach the bone, preparing the premetastatic niche. miRNAs from these exosomes change the balance RANKL/OPG that triggers osteoclast recruitment facilitating metastases by activation of the bone vicious circle. miR-21, overexpressed in bulk cells, the main cellular component of tumor, has the higher effect in modify microenvironment, being a potential therapeutic target. (FONDECYT 11121525 and 1140417). Citation Format: Eliana Andahur, Enrique A. Castellon, Chistian Ramos, Juan A. Fulla, Catherine A. Sanchez. miRNAs in exosomes from prostate cancer cells modify normal fibroblasts and osteoblasts favoring tumor spread and metastasis. [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 1562.
Cancer stem cells (CSCs) are related with cancer initiation, metastasis, resistance and recurrence, by regulating their environment in different ways, including the release of exosomes that modify surrounding and distant cells. Exosomes contain, among other molecules, miRNAs. By identifying the entire spectrum of miRNAs included in exosomes from prostate cancer (PCa) cells by next generation sequencing (NGS), is possible to identify miRNAs specifically released by CSCs, and those related with microenvironment regulation. We stablished adherent primary cell cultures (bulk cells) from PCa tissue from 5 patients. Later, from these cells we obtained CSCs enriched prostatospheres. From bulk and CSCs cultures we purified exosomes by precipitation (ExoQuick-TC, SBI) for miRNAs extraction (mirVana, Ambion). Quality of miRNAs was assessed by Bioanalizer (Agilent Technologies). NGS was performed in the Illumina platform (MiSeq, Illumina) according to manufacturer instructions. Bioinformatic analyses were carried out by Sistemas Genómicos. Overexpressed miRNAs were detected in plasma exosomes by TaqMan miRNA Assays (Lifetechnologies). We identified 1839 miRNAs in exosomes, and 223 species were differentially expressed between bulk and CSCs, but only 26 significantly. Nineteen of them were known species including 6 that were overexpressed in CSCs respect to bulk cells. These miRNAs target genes related with cell differentiation, matrix remodeling and genic expression. From all miRNA detected, 3 were highly overexpressed among all, and were detected in exosomes isolated from plasma of men with prostatic disease. Our study extensively characterizes the miRNA from exosomes of PCa cells. Exosomes contain high diversity of miRNAs, different in CSCs and bulk cells, suggesting differential regulation of their microenvironment. From these, CSCs exosome-related miRNAs could be released to bloodstream and play a role in PCa progression and metastasis (FONDECYT 11121525/1140417). Citation Format: Catherine A. Sanchez, Eliana Andahur, Rodrigo Valenzuela, Enrique Castellon, Christian Ramos, Juan Carlos Triviño. Identification of microRNAs from exosomes of bulk and stem cells from prostate cancer by next generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3990. doi:10.1158/1538-7445.AM2015-3990
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