Chemo-small extracellular vesicles released in cisplatin-resistance ovarian cancer cells are regulated by the lysosomal function.

Chemoresistance is a common problem in ovarian cancer (OvCa) treatment, where resistant cells, in response to chemotherapy, secrete small extracellular vesicles (sEVs), known as chemo-sEVs, that transfer resistance to recipient cells. sEVs are formed as intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs), whose trafficking is regulated by Ras-associated binding (RAB) GTPases that mediate sEVs secretion or lysosomal degradation. A decrease in lysosomal function can promote sEVs secretion, but the relationship between MVEs trafficking pathways and sEVs secretion in OvCa chemoresistance is unclear. Here, we show that A2780cis cisplatin (CCDP) resistant OvCa cells had an increased number of MVEs and ILVs structures, higher levels of Endosomal Sorting Complex Required for Transport (ESCRTs) machinery components, and RAB27A compared to A2780 CDDP-sensitive OvCa cells. CDDP promoted the secretion of chemo-sEVs in A2780cis cells, enriched in DNA damage response proteins. A2780cis cells exhibited poor lysosomal function with reduced levels of RAB7, essential in MVEs-Lysosomal trafficking. The silencing of RAB27A in A2780cis cells prevents the Chemo-EVs secretion, reduces its chemoresistance and restores lysosomal function and levels of RAB7, switching them into an A2780-like cellular phenotype. Enhancing lysosomal function with rapamycin reduced chemo-sEVs secretion. Our results suggest that adjusting the balance between secretory MVEs and lysosomal MVEs trafficking could be a promising strategy for overcoming CDDP chemoresistance in OvCa.

Tumor-derived hypoxic small extracellular vesicles promote endothelial cell migration and tube formation via ALS2/Rab5/ß-catenin signaling.

Tumor hypoxia has been associated with cancer progression, angiogenesis, and metastasis via modifications in the release and cargo composition of extracellular vesicles secreted by tumor cells. Indeed, hypoxic extracellular vesicles are known to trigger a variety of angiogenic responses via different mechanisms. We recently showed that hypoxia promotes endosomal signaling in tumor cells via HIF-1α-dependent induction of the guanine exchange factor ALS2, which activates Rab5, leading to downstream events involved in cell migration and invasion. Since Rab5-dependent signaling is required for endothelial cell migration and angiogenesis, we explored the possibility that hypoxia promotes the release of small extracellular vesicles containing ALS2, which in turn activate Rab5 in recipient endothelial cells leading to pro-angiogenic properties. In doing so, we found that hypoxia promoted ALS2 expression and incorporation as cargo within small extracellular vesicles, leading to subsequent transfer to recipient endothelial cells and promoting cell migration, tube formation, and downstream Rab5 activation. Consequently, ALS2-containing small extracellular vesicles increased early endosome size and number in recipient endothelial cells, which was followed by subsequent sequestration of components of the ß-catenin destruction complex within endosomal compartments, leading to stabilization and nuclear localization of ß-catenin. These events converged in the expression of ß-catenin target genes involved in angiogenesis. Knockdown of ALS2 in donor tumor cells precluded its incorporation into small extracellular vesicles, preventing Rab5-downstream events and endothelial cell responses, which depended on Rab5 activity and guanine exchange factor activity of ALS2. These findings indicate that vesicular ALS2, secreted in hypoxia, promotes endothelial cell events leading to angiogenesis. Finally, these events might explain how tumor angiogenesis proceeds in hypoxic conditions.

Connexin46 Expression Enhances Cancer Stem Cell and Epithelial-to-Mesenchymal Transition Characteristics of Human Breast Cancer MCF-7 Cells.

Connexins (Cxs) are a family of proteins that form two different types of ion channels: hemichannels and gap junction channels. These channels participate in cellular communication, enabling them to share information and act as a synchronized syncytium. This cellular communication has been considered a strong tumor suppressor, but it is now recognized that some type of Cxs can be pro-tumorigenic. For example, Cx46 expression is increased in human breast cancer samples and correlates with cancer stem cell (CSC) characteristics in human glioma. Thus, we explored whether Cx46 and glioma cells, can set up CSC and epithelial-to-mesenchymal transition (EMT) properties in a breast cancer cell line. To this end, we transfected MCF-7 cells with Cx46 attached to a green fluorescent protein (Cx46GFP), and we determined how its expression orchestrates both the gene-expression and functional changes associated with CSC and EMT. We observed that Cx46GFP increased Sox2, Nanog, and OCT4 mRNA levels associated with a high capacity to form monoclonal colonies and tumorspheres. Similarly, Cx46GFP increased the mRNA levels of n-cadherin, Vimentin, Snail and Zeb1 to a higher migratory and invasive capacity. Furthermore, Cx46GFP transfected in MCF-7 cells induced the release of higher amounts of VEGF, which promoted angiogenesis in HUVEC cells. We demonstrated for the first time that Cx46 modulates CSC and EMT properties in breast cancer cells and thus could be relevant in the design of future cancer therapies.

Connexin-46 Contained in Extracellular Vesicles Enhance Malignancy Features in Breast Cancer Cells.

Under normal conditions, almost all cell types communicate with their neighboring cells through gap junction channels (GJC), facilitating cellular and tissue homeostasis. A GJC is formed by the interaction of two hemichannels; each one of these hemichannels in turn is formed by six subunits of transmembrane proteins called connexins (Cx). For many years, it was believed that the loss of GJC-mediated intercellular communication was a hallmark in cancer development. However, nowadays this paradigm is changing. The connexin 46 (Cx46), which is almost exclusively expressed in the eye lens, is upregulated in human breast cancer, and is correlated with tumor growth in a Xenograft mouse model. On the other hand, extracellular vesicles (EVs) have an important role in long-distance communication under physiological conditions. In the last decade, EVs also have been recognized as key players in cancer aggressiveness. The aim of this work was to explore the involvement of Cx46 in EV-mediated intercellular communication. Here, we demonstrated for the first time, that Cx46 is contained in EVs released from breast cancer cells overexpressing Cx46 (EVs-Cx46). This EV-Cx46 facilitates the interaction between EVs and the recipient cell resulting in an increase in their migration and invasion properties. Our results suggest that EV-Cx46 could be a marker of cancer malignancy and open the possibility to consider Cx46 as a new therapeutic target in cancer treatment.

Environmental factors and management practices controlling oxygen dynamics in agricultural irrigation ponds in a semiarid Mediterranean region: implications for pond agricultural functions.

A water quality study was carried out on 40 irrigation ponds located within the main greenhouse areas on the Almería coast, placing special emphasis on the factors controlling the oxygen dynamics, a relevant aspect with agricultural and environmental implications. Considering chemical, physical and biological water characteristics, agricultural irrigation ponds were satisfactorily classified by cluster analysis in four groups. These were congruently arranged by principal components analysis along four main environmental gradients: trophic status, photosynthetic activity, water mineralisation and presence of submerged aquatic vegetation (SAV). Dissolved oxygen (DO) values differed highly among and within each of the four pond groups. DO dynamics was mainly depended on photosynthetic activity, and the environmental factors and management practices controlling it: seasonal and daily climatic changes, pond management (open vs. covered ponds and presence/absence of aquatic vegetation) and trophic status. Overall, different diurnal DO patterns were found between open and covered ponds. The former usually presented DO values above saturation and increasingly higher from early morning to mid-afternoon due to the photosynthetic activity of algae and macrophytic vegetation. In contrast, covered ponds showed relatively stable DO values during the diurnal period regardless of climatic conditions, with absolute values around or below saturation level. Globally, our results suggest that open ponds, with macrophytes concentrated in the deeper layer, can be an effective and sustainable management method of water oxygen enrichment.