In the last decade, substantial efforts have been made to identify NAD
biosynthesis inhibitors, specifically against nicotinamide phosphoribosyltransferase (NAMPT), as preclinical studies indicate ...their potential efficacy as cancer drugs. However, the clinical activity of NAMPT inhibitors has proven limited, suggesting that alternative NAD
production routes exploited by tumors confer resistance. Here, we show the gene encoding nicotinic acid phosphoribosyltransferase (NAPRT), a second NAD
-producing enzyme, is amplified and overexpressed in a subset of common types of cancer, including ovarian cancer, where NAPRT expression correlates with a BRCAness gene expression signature. Both NAPRT and NAMPT increased intracellular NAD
levels. NAPRT silencing reduced energy status, protein synthesis, and cell size in ovarian and pancreatic cancer cells. NAPRT silencing sensitized cells to NAMPT inhibitors both
and
; similar results were obtained with the NAPRT inhibitor 2-hydroxynicotinic acid. Reducing NAPRT levels in a BRCA2-deficient cancer cell line exacerbated DNA damage in response to chemotherapeutics. In conclusion, NAPRT-dependent NAD
biosynthesis contributes to cell metabolism and to the DNA repair process in a subset of tumors. This knowledge could be used to increase the efficacy of NAMPT inhibitors and chemotherapy.
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Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of ...soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived‐MSCs to investigate their involvement as modulators of MSC anti‐inflammatory effects inducing macrophage polarization. The EV‐isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo). Both types of EVs were efficiently internalized by responding bone marrow‐derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin‐induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV‐treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC‐EVs possess effective anti‐inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. Stem Cells Translational Medicine 2017 Stem Cells Translational Medicine 2017;6:1018–1028
Abstract Angiogenesis plays a central role in bone regeneration, not only for the transport of nutrients, but also for locally directing skeletal stem/progenitor cells. Following ectopic implantation ...of porous ceramic cubes seeded with mouse GFP-labeled mesenchymal stem cells (MSC) into syngenic mice, we investigated the cascade of events leading to bone formation. Implants harvested at different times were enzymatically digested to generate single-cell suspensions. Recovered cells were sorted to separate GFP + implanted MSC and host recruited GFP- cells. We isolated and characterized two different waves of cells, migrating from the host to the MSC-seeded ceramic. The first migrated cell population, recovered 7 days after implantation, was enriched in CD31 + endothelial progenitors, while the second one, recruited at day 11, was enriched in CD146 + pericyte-like cells. Both populations were not recruited into the scaffold following implantation of a non-MSC seeded ceramic. Pericyte-like cell mobilization was dependent on the first migrated endothelial cell population. Pericyte-like cells retained properties distinctive of stem cells, such as capacity of performing a high number of in vitro cell divisions and showed an osteogenic potential. Studies on the cross talk between implanted exogenous MSC and resident stem/progenitor cells could open new perspectives for future clinical applications.
The high invasive phenotype of glioblastoma is one of the main causes of therapy inefficacy and tumor relapse. Cell adhesion molecules of the cadherin family are involved in cell migration and are ...known as master regulators of epithelial tumor invasiveness, but their role in glioblastoma is less understood. In particular, we recently demonstrated, in the syngeneic murine model, the occurrence of a previously undescribed cadherin switch between Cdh2 and Cdh4 during gliomagenesis, which is necessary for the acquisition of the highly infiltrative and tumorigenic phenotype of these cells. In the present study, we tested the role of Cdh4 in human gliomas. Our results on patient-derived glioma cells demonstrate a positive correlation between Cdh4 expression levels and the loss of cell-cell contact inhibition of proliferation controls that allows cells to proliferate over confluence. Moreover, the silencing of Cdh4 by artificial microRNAs induced a decrease in the infiltrative ability of human glioma cells both in vitro and in vivo. More strikingly, Cdh4 silencing induced an impairment of the tumorigenic potential of these cells after orthotopic transplantation in immunodeficient mice. Overall, we conclude that in human glioblastoma, Cdh4 can also actively contribute in regulating cell invasiveness and malignancy.
The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine. Although adipose tissue‐and bone ...marrow‐derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). In this study, we carried out a detailed and comparative characterization of middle‐ and small‐sized EVs (mEVs and sEVs, respectively) released by either ADSCs or BMSCs. Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration, and marker expression, they exhibited different characteristics in terms of protein content and functional effects. ADSC‐EVs overexpressed pro‐angiogenic factors in comparison to the BMSC‐counterpart, and, consequently, they were able to induce a significant increase in endothelial cord outgrowth. On the other hand, BMSC‐EVs contained a higher amount of pro‐differentiation and chemotactic proteins, and they were able to prompt growth plate organization. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications.
This study shows how the differences among bone marrow‐ and adipose tissue‐derived mesenchymal stromal cells (BMSCs and ADSCs, respectively) are mirrored in the corresponding extracellular vesicles (EVs). The role of ADSCs‐ and BMSCs‐derived EVs in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics.
The study of circulating cancer-derived components (circulome) is considered the new frontier of liquid biopsy. Despite the recognized role of circulome biomarkers, their comparative molecular ...profiling is not yet routine. In advanced breast cancer (BC), approximately 40% of hormone-receptor-positive, HER2-negative BC cases harbor druggable
mutations suitable for combined alpelisib/fulvestrant treatment. This pilot study investigates
mutations in circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (EVs) with the aim of determining which information on molecular targetable profiling could be recollected in each of them. The in-depth molecular analysis of four BC patients demonstrated, as a proof-of-concept study, that it is possible to retrieve mutational information in the three components. Patient-specific
mutations were found in both tissue and ctDNA and in 3/4 cases, as well as in CTCs, in the classical population (large-sized CD45-/EpCAM+/- cells), and/or in the "non-conventional" sub-population (smaller-sized CD44+/EpCAM-/CD45- cells). Consistent mutational profiles of EVs with CTCs suggest that they may have been released by CTCs. This preliminary evidence on the molecular content of the different circulating biomaterials suggests their possible function as a mirror of the intrinsic heterogeneity of BC. Moreover, this study demonstrates, through mutational assessment, the tumor origin of the different CTC sub-populations sustaining the translational value of the circulome for a more comprehensive picture of the disease.
We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative ...paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were analyzed for protein and chemokine/cytokine content, and the EV cargo was further investigated by RNA sequencing. The phenotype of fetal- and perinatal hAFS, along with their corresponding secretome formulations, overlapped; yet, fetal hAFS showed immature oxidative phosphorylation activity when compared to perinatal ones. The profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. Both cell sources provided formulations enriched with neurotrophic, immunomodulatory, anti-fibrotic and endothelial stimulating factors, and the immature fetal hAFS secretome was defined by a more pronounced pro-vasculogenic, regenerative, pro-resolving and anti-aging profile. Small RNA profiling showed microRNA enrichment in both fetal- and perinatal hAFS-EV cargo, with a stably- expressed pro-resolving core as a reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.
Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in ...cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for "priming" MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.
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