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.

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.

Pharmacological Inhibition of p-21 Activated Kinase (PAK) Restores Impaired Neurite Outgrowth and Remodeling in a Cellular Model of Down Syndrome.

Down syndrome (DS) is characterized by the trisomy of chromosome 21 and by cognitive deficits that have been related to neuronal morphological alterations in humans, as well as in animal models. The gene encoding for amyloid precursor protein (APP) is present in autosome 21, and its overexpression in DS has been linked to neuronal dysfunction, cognitive deficit, and Alzheimer's disease-like dementia. In particular, the neuronal ability to extend processes and branching is affected. Current evidence suggests that APP could also regulate neurite growth through its role in the actin cytoskeleton, in part by influencing p21-activated kinase (PAK) activity. The latter effect is carried out by an increased abundance of the caspase cleavage-released carboxy-terminal C31 fragment. In this work, using a neuronal cell line named CTb, which derived from the cerebral cortex of a trisomy 16 mouse, an animal model of human DS, we observed an overexpression of APP, elevated caspase activity, augmented cleavage of the C-terminal fragment of APP, and increased PAK1 phosphorylation. Morphometric analyses showed that inhibition of PAK1 activity with FRAX486 increased the average length of the neurites, the number of crossings per Sholl ring, the formation of new processes, and stimulated the loss of processes. Considering our results, we propose that PAK hyperphosphorylation impairs neurite outgrowth and remodeling in the cellular model of DS, and therefore we suggest that PAK1 may be a potential pharmacological target.

Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

Role of the Pro-Inflammatory Tumor Microenvironment in Extracellular Vesicle-Mediated Transfer of Therapy Resistance.

Advances in our understanding of cancer biology have contributed to generating different treatments to improve the survival of cancer patients. However, although initially most of the therapies are effective, relapse and recurrence occur in a large percentage of these cases after the treatment, and patients then die subsequently due to the development of therapy resistance in residual cancer cells. A large spectrum of molecular and cellular mechanisms have been identified as important contributors to therapy resistance, and more recently the inflammatory tumor microenvironment (TME) has been ascribed an important function as a source of signals generated by the TME that modulate cellular processes in the tumor cells, such as to favor the acquisition of therapy resistance. Currently, extracellular vesicles (EVs) are considered one of the main means of communication between cells of the TME and have emerged as crucial modulators of cancer drug resistance. Important in this context is, also, the inflammatory TME that can be caused by several conditions, including hypoxia and following chemotherapy, among others. These inflammatory conditions modulate the release and composition of EVs within the TME, which in turn alters the responses of the tumor cells to cancer therapies. The TME has been ascribed an important function as a source of signals that modulate cellular processes in the tumor cells, such as to favor the acquisition of therapy resistance. Although generally the main cellular components considered to participate in generating a pro-inflammatory TME are from the immune system (for instance, macrophages), more recently other types of cells of the TME have also been shown to participate in this process, including adipocytes, cancer-associated fibroblasts, endothelial cells, cancer stem cells, as well as the tumor cells. In this review, we focus on summarizing available information relating to the impact of a pro-inflammatory tumor microenvironment on the release of EVs derived from both cancer cells and cells of the TME, and how these EVs contribute to resistance to cancer therapies.