Keeping an open mind: highlights and controversies of the breast cancer stem cell theory

The discovery that breast cancers contain stem-like cells has fuelled exciting research in the last few years. These cells are referred to as breast cancer stem cells (BCSCs) and are thought to be involved in tumor initiation, progression, and metastasis. Being intrinsically resistant to chemo- and radiotherapy, they are also considered responsible for recurrence of the disease after treatment. BCSCs have been suggested to be at the basis of tumor complexity, as they have the ability to self-renew and give rise to highly proliferating and terminally differentiated cancer cells that comprise the heterogeneous bulk of the tumor. There has been much speculation on the BCSC model, and in this review we address some fundamental questions, such as the identity of BCSCs and their involvement in tumor intra- and interheterogeneity. As an alternative to the BCSC model, we discuss clonal evolution, as both theories show extensive evidence in support of their arguments. Finally, we discuss a unifying idea that reconciles both models, which is based on stem cell plasticity and epigenetic modifications induced by the tumor microenvironment. The implications of cancer stem cell plasticity for drug discovery and future therapeutic interventions are presented

Crack of a helicopter main rotor actuator attachment: failure analysis and lessons learned

A Light Utility Helicopter (LUH), in the course of a training flight, leaving the ground during thetaxi to take off, went into an uncontrolled rolling to the right; consequently the helicopter gradually laid downon the right side. The impact with the runway destroyed the rotating blades up to the hubs rotor. The accidentinvestigation focused on main rotor oscillatory plate servo actuators . These components, directly linked to thecloche movements, regulate main rotor blades plane tilt and pitch. Following the preliminary examination, onlyfront servo actuator attachment was found to be broken in two parts. In detail, the present paper deals with thefracture analysis results. The servo actuator attachment material is a 2014 Aluminum alloy extrudate, undergoneto T651 heat treatment. Fracture surfaces were examined by optical and electronic microscopy in order todetermine the main morphological features and consequently to trace the origin of failure mechanism andcauses. The accordance with the specification requirements about alloy composition was verified by quantitativeelementary analysis through inductive coupled plasma spectroscopy (ICP); furthermore, semi-quantitativeelementary analysis was locally verified by Energy dispersion spectroscopy X ray (EDS_RX). Finally, thehydrogen content of the material was evaluated by the total hydrogen analysis. Microstructural andtechnological alloy characteristics were verified as well by using metallographic microscopy and hardness testingof the material.Macroscopic fracture surfaces evidences were characterized by the lack of any significant plastic deformationsand by the presence of symmetry compared to the servo actuator axis. Microscopic fracture features of both theinvestigated surfaces were not coherent to the hypothesis of an impact of the main rotor to the soil. Furtherachieved evidences, such as grain boundary fracture propagation, the presence of corrosion products, were all inaccordance with a Stress Corrosion Cracking (SCC) progressive fracture mechanism.Finite Element Analysis (FEA) located the highest tensile stress value, when the servo actuator is in its nominalworking condition, at the same points where the corrosion products were more concentrated (i.e. in the part ofthe fracture exposed to oxidative air effect for the longest time). The good agreement between FEA andmorphological evidences allowed to determine the progressive fracture origin area, though it was not possible toindividuate the crack initiation point. In fact, in correspondence to the initiation area of both the fracturesurfaces, shining and flat morphology was found;. then there were evidence of plastic deformations, due to thedetachment of a servo actuator part.The ICP analysis and hardness testing results were in accordance with the material specification requirements.However, the hydrogen content was one order of magnitude greater than the required value and many andunexpected globular formations were observed on the fracture surface. Part of these were dendritic formations,while the others looked smooth and shining. Further, FESEM boundary grain observation gave evidences of ahigh presence of precipitates on the investigated surfaces. Hence, observed microstructural characteristics,boundary grain precipitates and globular formations allowed to hypothesize possible overheating/eutecticmelting phenomena, occurred during manufacturing processes.As widely reported in literature, the AA 2014 alloy is one of the aluminum-copper-magnesium-silicon type,employing copper aluminide (CuAl2 ) as the primary precipitation-hardening agent. The need for a maximumCu phase dispersion in solid solution requires a heat treatment range with an upper limit (507°C) that is near tothe melting of the eutectics (510°C). Moreover, since the 1960s, AA2014 has been defined as sensitive to SCC.This condition is mainly related to the presence of coarse-grained and aligned CuAl2 precipitates. Thisarrangement is due to an overheating (more than 507°C) or to a cooling process carried out too slowly.Microstructural analysis was carried out on three items: 1) a large portion of the broken actuator attachment; 2)on a servoactuator coming from the same production batch; 3) on a servo actuator coming from a differentproduction batch.The microstructure from the broken actuator attachment showed a great amount of precipitates (secondphases) lengthwise aligned to the boundary grain, pores, and also cavities and dendritic globular formations.Analysis results, morphology evidences and reference images available on scientific literature were found to bein excellent agreement and validated the embrittlement and subsequent SCC mechanism hypotesis(intergranular failure propagation).In conclusion, flight accident causes are attributable to main rotor actuator attachment failure.Failure mechanism is classifiable as SCC supported by microstructural anomalies of the material. Theinvestigation of the manufacturing process highlighted how one of the servo actuator batches was not properlyproduced due to poor control and accuracy of heat treatment temperature and/or cooling time. This led tohydrogen embrittlement and to a microstructural problem (globular formations and boundary grainprecipitates). The combination of those phenomena caused an increase of the SCC sensitivity and were thebasic progressive failure driving forces.Nevertheless, as above mentioned, alloy composition was found compliant with the material specificationrequirements and this just because none of the scheduled quality control tests is able to determine the peculiarmicrostructural anomalies reported

Dolichopoda cave crickets from Peloponnese (Orthoptera, Rhaphidophoridae): molecular and morphological investigations reveal four new species for Greece

Three species belonging to the genus Dolichopoda (Orthoptera; Rhaphidopohoridae) are known so far from the Peloponnese, all endemic to the area. In particular, D. matsakisi is known from two mountains in the North, while D. dalensi is present in the east region. The third species, D. unicolor, is distributed in the southern part of the Peloponnese, inhabiting caves on Mt Taygetos and Mani Peninsula. Recently, extensive sampling work in most of the Peloponnese has led to the discovery of new taxa, morphologically differentiated by the above three known species. To investigate the delimitation of the Peloponnesian species of Dolichopoda, we performed both morphological and molecular analyses. Morphological analysis was carried out by considering diagnostic characters generally used to distinguish different taxa, as the shape of epiphallus in males and the subgenital plate in females. Molecular analysis was performed by sequencing three mitochondrial genes, 12S rRNA, 16S rRNA, and COI, and one nuclear gene, 28S rRNA. Results from both morphological and molecular analyses were used to revise the taxonomic arrangement of the Peloponnesian species. On the whole, we were able to distinguish seven lineages of Peloponnesian Dolichopoda species, of which D. kofinasi n.sp., D.epidavrii n.sp., D. poseidonica n.sp., and D. propanti n.sp. are described as new species

Failure of cargo aileron’s actuator

During a ferry flight, in a standard operation condition and at cruising level, a military cargo experienced a double hydraulic system failure due to a structural damage of the dual booster actuator. The booster actuator is the main component in mechanism of aileron’s deflection. The crew was able to arrange an emergency landing thanks to the spare oil onboard: load specialists refilled the hydraulic reservoirs. Due to safety concerns and in order to prevent the possibility of other similar incidents, a technical investigation took place. The study aimed to carry out the analysis of root causes of the actuator failure. The Booster actuator is composed mainly by the piston rod and its aluminum external case (AA7049). The assembly has two bronze caps on both ends. These are fixed in position by means of two retainers. At one end of the actuator case is placed a trunnion: a cylindrical protrusion used as a pivoting point on the aircraft. The fracture was located at one end of the case, on the trunnion side, in correspondence to the cap and over the retainer. One of the two fracture surfaces was found separated to the case and with the cap entangled inside. The fracture surfaces of the external case indicated fatigue crack growth followed by ductile separation. The failure analysis was performed by means of optical, metallographic, digital and electronic microscopy. The collected evidences showed a multiple initiation fracture mechanism. Moreover, 3D scanner reconstruction and numerical simulation demonstrated that dimensional non conformances and thermal loads caused an abnormal stress concentration.Stress concentration was located along the case assy outer surface where the fatigue crack originated. The progressive rupture mechanism grew under cyclical axial load due to the normal operations. Recommendationswere issued in order to improve dimensional controls and assembly procedures during production and  overhaul activities

The diet of Weddell seals (Leptonychotes weddellii) in Terra Nova Bay using stable isotope analysis

Stable isotope analyses were used to investigate the diet of Weddell seals in Terra Nova Bay (Ross Sea) and the potential variation of their foraging behaviour with age, sex and body mass. For this purpose, skin samples were collected from adult breeding seals and pups, together with muscle samples of their potential prey. Our results showed variation in foraging behavior between age classes, with pups reporting lower δ13C values than adults, while no significant differences in δ15N were recorded. In addition, contrary to expectations, a mixing model analysis showed that adult seals foraged mainly on shallow benthic prey, such as Trematomus spp. (34.1%) and Dissostichus mawsoni (21.1%), rather than on pelagic fish, such as Pleuragramma antarcticum (9.8%). Overall, with this paper we provide novel diet information on a seal colony not previously sampled, adding new insight into the feeding ecology of a top Antarctic predator

HOXC8 regulates self-renewal, differentiation and transformation of breast cancer stem cells

Background: Homeobox genes are master regulators of cell fate during embryonic development and their expression is altered in cancer. By regulating the balance between cell proliferation and differentiation, they maintain homeostasis of normal tissues. Here, we screened the expression of homeobox genes in mammary stem cells to establish their role in stem cells transformation in breast cancer. Methods: Using a Homeobox Genes PCR array, we screened 83 homeobox genes in normal cancer breast stem/progenitor cells isolated by flow cytometry. The candidate gene HOXC8 epigenetic regulation was studied by DNA methylation and miRNA expression analyses. Self-renewal and differentiation of HOXC8-overexpressing or knockdown cells were assessed by flow cytometry and mammosphere, 3D matrigel and soft agar assays. Clinical relevance of in vitro findings were validated by bioinformatics analysis of patient datasets from TCGA and METABRIC studies. Results: In this study we demonstrate altered expression of homeobox genes in breast cancer stem/progenitor cells. HOXC8 was consistently downregulated in stem/progenitor cells of all breast molecular subtypes, thus representing an interesting tumour suppressor candidate. We show that downregulated expression of HOXC8 is associated with DNA methylation at the gene promoter and expression of miR196 family members. Functional studies demonstrated that HOXC8 gain of function induces a decrease in the CD44+/CD24-/low cancer stem cell population and proportion of chemoresistant cells, with a concomitant increase in CD24+ differentiated cells. Increased HOXC8 levels also decrease the ability of cancer cells to form mammospheres and to grow in anchorage-independent conditions. Furthermore, loss of HOXC8 in non-tumorigenic mammary epithelial cells expands the cancer stem/progenitor cells pool, increases stem cell self-renewal, prevents differentiation induced by retinoic acid and induces a transformed phenotype. Conclusions: Taken together, our study points to an important role of homeobox genes in breast cancer stem/progenitor cell function and establishes HOXC8 as a suppressor of stemness and transformation in the mammary gland lineag

Regulation of vascular endothelial growth factor in prostate cancer

Prostate cancer (PCa) is the most common malignancy affecting men in the western world. Although radical prostatectomy and radiation therapy can successfully treat PCa in the majority of patients, up to ∼30% will experience local recurrence or metastatic disease. Prostate carcinogenesis and progression is typically an androgen-dependent process. For this reason, therapies for recurrent PCa target androgen biosynthesis and androgen receptor function. Such androgen deprivation therapies (ADT) are effective initially, but the duration of response is typically ≤24 months. Although ADT and taxane-based chemotherapy have delivered survival benefits, metastatic PCa remains incurable. Therefore, it is essential to establish the cellular and molecular mechanisms that enable localized PCas to invade and disseminate. It has long been accepted that metastases require angiogenesis. In the present review, we examine the essential role for angiogenesis in PCa metastases, and we focus in particular on the current understanding of the regulation of vascular endothelial growth factor (VEGF) in localized and metastatic PCa. We highlight recent advances in understanding the role of VEGF in regulating the interaction of cancer cells with tumor-associated immune cells during the metastatic process of PCa. We summarize the established mechanisms of transcriptional and post-transcriptional regulation of VEGF in PCa cells and outline the molecular insights obtained from preclinical animal models of PCa. Finally, we summarize the current state of anti-angiogenesis therapies for PCa and consider how existing therapies impact VEGF signaling

A 3D Heterotypic Breast Cancer Model Demonstrates a Role for Mesenchymal Stem Cells in Driving a Proliferative and Invasive Phenotype

Previous indirect 2D co-culture studies have demonstrated that mesenchymal stem cells (MSCs) promote breast cancer (BC) progression through secretion of paracrine factors including growth factors, cytokines and chemokines. In order to investigate this aspect of the tumour microenvironment in a more relevant 3D co-culture model, spheroids incorporating breast cancer cells (BCCs), both cell lines and primary BCCs expanded as patient-derived xenografts, and MSCs were established. MSCs in co-cultures were shown to enhance proliferation of estrogen receptor (ER)/progesterone receptor (PR)-positive BCCs. In addition, co-culture resulted in downregulation of E-cadherin in parallel with upregulation of the epithelial-mesenchymal transition (EMT)-relation transcription factor, SNAIL. Cytoplasmic relocalization of ski-related novel protein N (SnON), a negative regulator of transforming growth factor-beta (TGF-β) signalling, and of β-catenin, involved in a number of pathways including Wnt signalling, was also observed in BCCs in co-cultures in contrast to monocultures. In addition, the β-catenin inhibitor, 3-[[(4-methylphenyl)sulfonyl]amino]-benzoic acid methyl ester (MSAB), mediated reduced growth and invasion in the co-cultures. This study highlights the potential role for SnON as a biomarker for BC invasiveness, and the importance of interactions between TGF-β and Wnt signalling, involving SnON. Such pathways may contribute towards identifying possible targets for therapeutic intervention in BC patients

Cyclin A1 and P450 aromatase promote metastatic homing and growth of stem-like prostate cancer cells in the bone marrow

Bone metastasis is a leading cause of morbidity and mortality in prostate cancer (PCa). While cancer stem-like cells have been implicated as a cell of origin for PCa metastases, the pathways which enable metastatic development at distal sites remain largely unknown. In this study, we illuminate pathways relevant to bone metastasis in this disease. We observed that cyclin A1 (CCNA1) protein expression was relatively higher in PCa metastatic lesions in lymph node, lung, and bone/bone marrow. In both primary and metastatic tissues, cyclin A1 expression was also correlated with aromatase (CYP19A1), a key enzyme that directly regulates the local balance of androgens to estrogens. Cyclin A1 overexpression in the stem-like ALDHhigh subpopulation of PC3M cells, one model of PCa, enabled bone marrow integration and metastatic growth. Further, cells obtained from bone marrow metastatic lesions displayed self-renewal capability in colony forming assays. In the bone marrow, Cyclin A1 and aromatase enhanced local bone marrow-releasing factors, including androgen receptor, estrogen and matrix metalloproteinase MMP9 and promoted hte metastatic growth of PCa cells. Moreover, ALDHhigh tumor cells expressing elevated levels of aromatase stimulated tumor/host estrogen production and acquired a growth advantage in the presence of host bone marrow cells. Overall, these findings suggest that local production of steroids and MMPs in the bone marrow may provide a suitable microenvironment for ALDHhigh PCa cells to establish metastatic growths, offering new approaches to therapeutically target bone metastases