Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed

Diclofenac-Induced Apoptosis in the Neuroblastoma Cell Line SH-SY5Y: Possible Involvement of the Mitochondrial Superoxide Dismutase

Diclofenac, a nonsteroidal anti-inflammatory drug, induces apoptosis on the neuroblastoma cell line SH-SY5Y through a mitochondrial dysfunction, affecting some antioxidant mechanisms. Indeed, the time- and dose-dependent increase of apoptosis is associated to an early enhancement of the reactive oxygen species (ROS). Mitochondrial superoxide dismutase (SOD2) plays a crucial role in the defence against ROS, thus protecting against several apoptotic stimuli. Diclofenac decreased the protein levels and the enzymatic activity of SOD2, without any significant impairment of the corresponding mRNA levels in the SH-SY5Y extracts. When cells were incubated with an archaeal exogenous thioredoxin, an attenuation of the diclofenac-induced apoptosis was observed, together with an increase of SOD2 protein levels. Furthermore, diclofenac impaired the mitochondrial membrane potential, leading to a release of cytochrome c. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis of SH-SY5Y cells and point to a possible role of SOD2 in this process

Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5?9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 ?M. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds

Generation of spheroids from human primary myofibroblasts: an experimental system to study myofibroblasts deactivation

Fibroblasts represent a heterogeneous cell population, that in adult body maintains the homeostasis of the extracellular matrix (ECM) and can acquire an immunoregulatory phenotype. Indeed, activated fibroblasts produce large amounts of cyclooxygenase-2 (COX-2) and proinflammatory cytokines (1). The activation of fibroblasts is represented by their differentiation into myofibroblasts. This process, either in wound healing or cancer tissue, is associated with the expression of alpha-smooth muscle actin (alpha-SMA), increased levels of growth factors and ECM-degrading proteases (2). Moreover, myofibroblasts form clusters in wound healing process and hypertrophic scars. In particular, cell clusters of hypertrophic scars are represented by nodules of myofibroblasts (3). It is known that human dermal fibroblasts established from neonatal foreskin, and forced in vitro to form clusters named spheroids, are activated to produce massive amounts of COX-2, prostaglandins and proinflammatory cytokines: this process leads to a programmed necrosis, designated “nemosis” (1). In the present study we generated spheroids from human primary myofibroblasts of skin, to evaluate necrotic, inflammation and activation markers during myofibroblasts clustering. Western blotting analysis, showing low levels of COX-2 and a significant decrease of alpha-SMA in protein extracts of spheroids, led to hypothesize that myofibroblasts have undergone a deactivation process within spheroids. This hypothesis is confirmed by cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines, by confocal immunofluorescence analysis of connexin 43 and immunohistochemical evaluation of proliferation marker Ki-67. This work could represent an experimental model to study myofibroblasts deactivation and highlights an alternative process regulating the turnover of myofibroblasts

Analysis of SOD3 and Akt in ascending aortic aneurysm

Ascending aortic aneurysm (AsAA) is divided into three different forms: syndromic, familial non-syndromic, and degenerative. Bicuspid aortic valve (BAV), occuring in 2% of the population, is the most frequent cardiac congenital abnormality, associated to AsAA. All the different forms of AsAAs are a consequence of cystic medial necrosis (CMN), characterized by apoptotic loss of smooth muscle cells (SMCs), fragmentation of elastic and collagen fibers and increased accumulation of mucoid material. The extracellular superoxide dismutase (SOD3) is a Cu/Zn enzyme, affecting redox state and homeostasis of extracellular matrix (ECM) (1). Moreover, the outsidein signalling from ECM modulates intracellular pathways regulating many cellular functions. The multifunctional Akt pathway affects survival and cellular proliferation and has important effects on the cardiovascular function. In this study we examined the relevance of SOD3 and Akt in AsAA pathogenesis. To this aim, the SOD3 and Akt protein levels were evaluated in normal ascending aortic tissues (n=6) and in tissues from AsAAs associated both to tricuspid aortic valve (TAV) (n=6) and BAV (n=6); moreover, we measured SOD3 activity in sera from healthy donors and patients with AsAA. Our data showed a reduction of SOD3 and phospho-Akt (pAkt) protein levels in AsAAs from BAV patients compared to normal donors; on the other hand, no differences emerged in SOD3 activity. Furthermore, immunohistochemical analysis performed on normal and pathological ascending aortic tissues showed a SOD3 immunostaining in both extracellular space and tunica media cells from normal ascending aortic tissues; conversely, no SOD3 immunostaining was detected in AsAAs tissues from both TAV and BAV patients. Our data show that SOD3 and pAkt could be associated to AsAA pathogenesis and suggest a link between ECM homeostasis and Akt survival pathway

Thyroid Cancer and Fibroblasts

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells

A pan-European epidemiological study reveals honey bee colony survival depends on beekeeper education and disease control

Reports of honey bee population decline has spurred many national efforts to understand the extent of the problem and to identify causative or associated factors. However, our collective understanding of the factors has been hampered by a lack of joined up trans-national effort. Moreover, the impacts of beekeeper knowledge and beekeeping management practices have often been overlooked, despite honey bees being a managed pollinator. Here, we established a standardised active monitoring network for 5 798 apiaries over two consecutive years to quantify honey bee colony mortality across 17 European countries. Our data demonstrate that overwinter losses ranged between 2% and 32%, and that high summer losses were likely to follow high winter losses. Multivariate Poisson regression models revealed that hobbyist beekeepers with small apiaries and little experience in beekeeping had double the winter mortality rate when compared to professional beekeepers. Furthermore, honey bees kept by professional beekeepers never showed signs of disease, unlike apiaries from hobbyist beekeepers that had symptoms of bacterial infection and heavy Varroa infestation. Our data highlight beekeeper background and apicultural practices as major drivers of honey bee colony losses. The benefits of conducting trans-national monitoring schemes and improving beekeeper training are discussed