Extracellular Vesicles Derived from Acidified Metastatic Melanoma Cells Stimulate Growth, Migration, and Stemness of Normal Keratinocytes

Abstract

Metastatic melanoma is a highly malignant tumor. Melanoma cells release extracellular vesicles (EVs), which contribute to the growth, metastasis, and malignancy of neighboring cells by transfer of tumor-promoting miRNAs, mRNA, and proteins. Melanoma microenvironment acidification promotes tumor progression and determines EVs’ properties. We studied the influence of EVs derived from metastatic melanoma cells cultivated at acidic (6.5) and normal (7.4) pH on the morphology and homeostasis of normal keratinocytes. Acidification of metastatic melanoma environment made EVs more prooncogenic with increased expression of prooncogenic mi221 RNA, stemless factor CD133, and pro-migration factor SNAI1, as well as with downregulated antitumor mir7 RNA. Incubation with EVs stimulated growth and migration both of metastatic melanoma cells and keratinocytes and changed the morphology of keratinocytes to stem-like phenotype, which was confirmed by increased expression of the stemness factors KLF and CD133. Activation of the AKT/mTOR and ERK signaling pathways and increased expression of epidermal growth factor receptor EGFR and SNAI1 were detected in keratinocytes upon incubation with EVs. Moreover, EVs reduced the production of different cytokines (IL6, IL10, and IL12) and adhesion factors (sICAM-1, sICAM-3, sPecam-1, and sCD40L) usually secreted by keratinocytes to control melanoma progression. Bioinformatic analysis revealed the correlation between decreased expression of these secreted factors and worse survival prognosis for patients with metastatic melanoma. Altogether, our data mean that metastatic melanoma EVs are important players in the transformation of normal keratinocytes.

miR-214-Enriched Extracellular Vesicles Released by Acid-Adapted Melanoma Cells Promote Inflammatory Macrophage-Dependent Tumor Trans-Endothelial Migration

“…Aware of the importance of translating this study in vivo, we plan to validate our results by using a model of experimental metastasis in syngeneic mice. In line with our observations, very recently, Bychkov and colleagues demonstrated that extracellular acidosis, via inducing metastatic melanoma cells to release EV with advanced pro-tumoral activity, sustained the acquisition of an increased malignant phenotype of melanoma cells themselves, also redirecting keratinocytes towards pro-tumoral activities [ 45 ]. This preclinical in vitro study allowed us to identify another mechanism through which the acidic TME may foster tumor progression and dissemination, exploiting the great potential of EV to convey pro-tumoral molecular signals capable of altering the microenvironment of secondary locations and prepare for metastatic colonization.…”

Contribution of Tumor-Derived Extracellular Vesicles to Malignant Transformation of Normal Cells

“…For example, incubation of keratinocytes with EVs derived from metastatic melanoma cells activates the AKT/mammalian target of rapamycin (mTOR) and extracellular-signal-regulated kinase (ERK) signaling pathways, thereby increasing cell migration ability. In addition, tumor-cell-derived EV-treated keratinocytes had a stem-like phenotype and increased expression of SNAI1 , KLF , and CD133 factors that trigger and maintain EMT [ 34 ]. Migration-enhancing morphological changes—namely, an increase in cell length and the number of filopodia—were also observed in normal lung epithelium BEAS-2B cells after the addition of exosomes from non-small lung carcinoma cell line H1299 [ 35 ].…”

Extracellular Vesicles Derived from Metastatic Melanoma Cells Transfer α7-nAChR mRNA, Thus Increasing the Surface Expression of the Receptor and Stimulating the Growth of Normal Keratinocytes

“…Для удаления эндогенных экзосом эмбриональную телячью сыворотку центрифугировали (70 мин, 120000 g), после чего фильтровали и смешивали со средой. Кератиноциты человека Het-1A (ATCC, США) культивировали в среде BEBM (Lonza, Швейцария) как описано ранее [7]. Внеклеточные везикулы выделяли из клеток метастатической меланомы как в [7]: клетки выращивали в среде без экзосом, культуральную среду центрифугировали последовательно при 10000 g (15 мин, 4°C) и при 120000 g (70 мин, 4°С).…”

“…Кератиноциты человека Het-1A (ATCC, США) культивировали в среде BEBM (Lonza, Швейцария) как описано ранее [7]. Внеклеточные везикулы выделяли из клеток метастатической меланомы как в [7]: клетки выращивали в среде без экзосом, культуральную среду центрифугировали последовательно при 10000 g (15 мин, 4°C) и при 120000 g (70 мин, 4°С). Белковые комплексы удаляли с помощью гель-фильтрации, используя сорбент Superdex G-250 (GE Healthcare, США).…”

“…Ранее мы показали, что внеклеточные везикулы, секретируемые клетками метастатических меланом, стимулируют рост, миграцию и стволовость нормальных кератиноцитов [7]. Процессы дифференцировки и роста нормальных кератиноцитов регулируются с участием никотинового рецептора ацетилхолина типа α7 (α7-nAChR) [8], активация которого никотином или его производными, содержащимися в табаке (нитрозаминами), приводит к злокачественной трансформации кератиноцитов [9].…”