In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.
PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9⌬C-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9⌬C bound to EGF(AB) H306Y , a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB) H306Y -PCSK9 interaction is pH-independent, LDLR H306Y binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9⌬C containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) 4 has recently emerged as a major regulator of low density lipoprotein (LDL) cholesterol levels in plasma and consequently as an important determinant of cardiovascular health in humans. The link between cardiovascular disease and PCSK9 was initially made following the discovery that the PCSK9 missense mutations, S127R and F216L (1), and later, D374Y (2), are associated with a form of autosomal dominant hypercholesterolemia, a risk factor for coronary heart disease. Subsequently, two PCSK9 nonsense mutations (Y142X and C679X) (3) and several missense mutations (R46L, G106R, N157K, G236S, R237W, L253F, N354I and A443T) (4 -6) have been found to be associated with hypocholesterolemia. Remarkable degrees of protection against coronary heart disease were observed in humans heterozygous for the mutations Y142X and C679X (88% reduced incidence) and by R46L (47% reduced incidence) (7). Consequently, PCSK9 represents a novel therapeutic target for the prevention of premature atherosclerosis and coronary heart disease, and an understanding of its mechanism of action is of significant medical interest.PCSK9 is the ninth member of the mammalian proprotein convertase family of serine proteases. The translated proprotein includes an N-terminal signal sequence directing its secretion (residues 1-30), a prodomain (residues 31-152), a catalytic domain (residues 153-451)...
Recent evidences suggest that stearoyl-CoA-desaturase 1 (SCD1), the enzyme involved in monounsaturated fatty acids synthesis, has a role in several cancers. We previously demonstrated that SCD1 is important in lung cancer stem cells survival and propagation. In this article, we first show, using primary cell cultures from human lung adenocarcinoma, that the effectors of the Hippo pathway, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), are required for the generation of lung cancer three-dimensional cultures and that SCD1 knock down and pharmacological inhibition both decrease expression, nuclear localization and transcriptional activity of YAP and TAZ. Regulation of YAP/TAZ by SCD1 is at least in part dependent upon β-catenin pathway activity, as YAP/TAZ downregulation induced by SCD1 blockade can be rescued by the addition of exogenous wnt3a ligand. In addition, SCD1 activation of nuclear YAP/TAZ requires inactivation of the β-catenin destruction complex. In line with the in vitro findings, immunohistochemistry analysis of lung adenocarcinoma samples showed that expression levels of SCD1 co-vary with those of β-catenin and YAP/TAZ. Mining available gene expression data sets allowed to observe that high co-expression levels of SCD1, β-catenin, YAP/TAZ and downstream targets have a strong negative prognostic value in lung adenocarcinoma. Finally, bioinformatics analyses directed to identify which gene combinations had synergistic effects on clinical outcome in lung cancer showed that poor survival is associated with high co-expression of SCD1, β-catenin and the YAP/TAZ downstream target birc5. In summary, our data demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway in lung cancer, and point to fatty acids metabolism as a key regulator of lung cancer stem cells.
Cancer stem cells (CSCs) are an uncommon subset of tumor cells capable of self-renewal, differentiating, and recreating the parental tumor when transplanted into the murine background. Over the past two decades, efforts toward understanding CSC biology culminated into identifying a set of signaling pathways sustaining "stemness". Nevertheless, while metabolic rewiring is nowadays considered a hallmark of cancer, no consensus has been reached on the metabolic features underlying the plastic nature of CSCs, which are capable of residing in a dormant state, and able to rapidly proliferate when the need to repopulate the tumor mass arises. An emerging concept in the field of CSC metabolism is that these cells are extremely reliant on the activity of enzymes involved in lipid metabolism, such as stearoyl-CoA desaturase 1 (SCD1) and 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR). Indeed, SCD1 and HMG-CoAR have been described as key factors for the correct function of a number of concatenated pathways involved in CSC fate decision, such as Hippo and Wnt. In the present review, we describe metabolic futures of CSCs with a special focus on lipid metabolism, which until now represents an underappreciated force in maintaining CSCs and an attractive therapeutic target.
Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.
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