Prostate cancer (PCa) pathogenesis relies on intercellular communication, which can involve tunneling nanotubes (TNTs) and extracellular vesicles (EVs). TNTs and EVs have been reported to transfer critical cargo involved in cellular function and signalling, prompting us to investigate the extent of organelle and protein transfer in PCa cells and the potential involvement of the androgen receptor. Using live cell imaging microscopy, we observed extensive formation of TNTs and EVs operating between PCa, non-malignant and immune cells. PCa cells were capable of transferring lysosomes, mitochondria, lipids and endoplasmic reticulum as well as syndecan-1, sortilin, Glut1 and Glut4. In mechanistic studies, androgen sensitive PCa cells exhibited changes in cell morphology when stimulated by R1881 treatment. Overexpression assays of a newly designed androgen receptor (AR) plasmid revealed its novel localization in PCa cellular vesicles, which were also transferred to neighbouring cells. Selected molecular machinery, thought to be involved in intercellular communication, was investigated by knockdown studies and Western blotting/immunofluorescence/scanning electron microscopy (SEM). PCa TNTs and EVs transported proteins and organelles, which may contain specialist signalling, programming and energy requirements that support cancer growth and progression. This makes these important intercellular communication systems ideal potential targets for therapeutic intervention.

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies on their therapeutic value in the lung EMT process are lacking. Bronchial epithelial (BEAS-2B, primary HAE cells) were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers collagen Ӏ (mRNA, protein) were analyzed. St-Ht31 disrupted AKAP-PKA interactions. TGF-β1 release was measured by ELISA. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin but increased collagen Ӏ and cell migration, a process reversed by PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95 and Yotiao. St-Ht31 decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration and reduced E-cadherin expression, a process blocked by TGF-β1 neutralizing antibody. Silencing of Ezrin, AKAP95 and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95 and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members define the ability of fenoterol, rolipram and cilostamide to modulate the EMT process, and are potential relevant targets in the treatment of COPD.

Rationale: Astroglial cells (astrocytes), previously considered mere support cells in the central nervous system, are now understood to be key players in integrating neuronal inputs and modulating synaptic density. Astrocytic processes surround the perikaryons and neuronal processes including dendritic spines and by extension or retraction, respectively, lyse or facilitate increases in number or size of synapses. Astrocytes express estrogen receptors and in animal models, including monkeys, have been shown to respond to estradiol by changing the disposition of their processes in a manner opposite to the number of synapses. Experimental: Our objective was to examine the effects of estradiol and two other clinically available Selective Estrogen Receptor Modulators (SERMs; tamoxifen and raloxifene) on astrocyte processes in human brain tissue. To accomplish this goal, we used simultaneous immunostaining of the glial cytoskeletal marker glial acidic protein (GFAP) and ezrin, a membrane-cytoskeletal linker, plus light microscopy image analysis to study the thinness and branching of randomly selected human temporal lobe neocortical astrocytes after a 60-minute incubation of 400µ temporal lobe slices. Effects of estradiol (10 nM), raloxifene and tamoxifen (1.0 µM) were compared with untreated control slices. Results: Changes were confirmed in the branchings of temporal lobe astrocytes during the 60-minute incubation period. Astrocytes in control slices extended their branches and became thinner. Parallel changes were present in tamoxifen-cultured slices. Raloxifene-treated slices showed significant reduction of astrocyte branching and thinning compared to controls (p = 0.01, 0.01, 0.06 for primary, secondary, and tertiary processes, respectively). An intermediate reduction of astroglial process size was present following estradiol treatment, but not at a statistically significant level compared to controls. Conclusions: These preliminary results support estrogen receptor modulator regulation of human astroglial processes in a manner consistent with previous reports on animals in which changes in glial spreading plays a role in synaptic changes. Neuroendocrinological implications are discussed.