Recurrent and multiple bladder tumors show conserved expression profiles

<p>Abstract</p> <p>Background</p> <p>Urothelial carcinomas originate from the epithelial cells of the inner lining of the bladder and may appear as single or as multiple synchronous tumors. Patients with urothelial carcinomas frequently show recurrences after treatment making follow-up necessary. The leading hypothesis explaining the origin of meta- and synchronous tumors assumes a monoclonal origin. However, the genetic relationship among consecutive tumors has been shown to be complex in as much as the genetic evolution does not adhere to the chronological appearance of the metachronous tumors. Consequently, genetically less evolved tumors may appear chronologically later than genetically related but more evolved tumors.</p> <p>Methods</p> <p>Forty-nine meta- or synchronous urothelial tumors from 22 patients were analyzed using expression profiling, conventional CGH, LOH, and mutation analyses.</p> <p>Results</p> <p>We show by CGH that partial chromosomal losses in the initial tumors may not be present in the recurring tumors, by LOH that different haplotypes may be lost and that detected regions of LOH may be smaller in recurring tumors, and that mutations present in the initial tumor may not be present in the recurring ones. In contrast we show that despite apparent genomic differences, the recurrent and multiple bladder tumors from the same patients display remarkably similar expression profiles.</p> <p>Conclusion</p> <p>Our findings show that even though the vast majority of the analyzed meta- and synchronous tumors from the same patients are not likely to have originated directly from the preceding tumor they still show remarkably similar expressions profiles. The presented data suggests that an expression profile is established early in tumor development and that this profile is stable and maintained in recurring tumors.</p

HAMLET Binding to α-Actinin Facilitates Tumor Cell Detachment

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed

Changes in Proteasome Structure and Function Caused by HAMLET in Tumor Cells

BACKGROUND: Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. METHODOLOGY/PRINCIPAL FINDINGS: HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. CONCLUSIONS/SIGNIFICANCE: The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells

Mechanisms of HAMLET-induced cancer cell death

HAMLET, a complex of α-lactalbumin and oleic acid, preferentially kills cancer cells and is also effective in vivo. HAMLET causes apoptosis but cells die even if this pathway is inhibited. Thus, the role of autophagy, an alternative cell death pathway, was examined. During autophagy double membrane-enclosed autophagosomes form around cellular material and deliver it to the lysosomes for degradation. After HAMLET treatment, such vesicles were observed and HAMLET also increased granular LC3-GFP staining confirming autophagosome accumulation. In addition, the autophagic form of LC3 increased if lysosomal degradation was inhibited, indicating a true increase in autophagic flux. Several factors that may activate autophagy were identified, including mitochondrial damage, inhibition of the autophagy inhibitor mTOR and increased the expression of autophagy components. Inhibition of autophagy by Atg5 and Beclin-1 siRNAs reduced cell death suggesting that autophagy is an important part of the cell death programme. As autophagy can be initiated by metabolic stress and as metabolism is altered in cancer cells we investigated if HAMLET-induced cell death involves an effect on metabolism. Healthy cells, which were less sensitive to HAMLET, had lower levels of c-Myc, an important regulator of glucose metabolism, and knockdown of c-Myc in cancer cells reduced their sensitivity. In contrast, cancer cell death was enhanced by glycolysis inhibition. HAMLET was also shown to bind the glycolytic enzyme hexokinase 1 (HK1) and to reduce HK activity, ATP levels and lactate release. Also, knockdown of HK1 and the glycolysis-enhancing HIF1A sensitized cancer cells to HAMLET whereas knockdown of the glycolysis-regulating protein PFKFB1 reduced sensitivity. Finally, mass spectrometry-based analysis indicated that HAMLET has a broad impact on metabolism possibly due to binding of HK1. In an additional screen for HAMLET-binding proteins prohibitin-2, a multifunctional protein found at the same cellular sites as HAMLET, was identified. The binding was confirmed in vitro and HAMLET was shown to also bind to the related prohibitin. Co-staining for HAMLET and prohibitins revealed colocalization in the cytoplasm. In addition, nuclear staining for both prohibitins was reduced, as described in camptothecin-induced cancer cell death. Furthermore, prohibitin-2 siRNA reduced cell death after HAMLET treatment suggesting that prohibitins may play a role in HAMLET-induced cell death. Besides cell death, HAMLET also causes cancer cell detachment and α-actinin-4, which crosslinks the actin cytoskeleton and focal adhesions, was identified in a screen as a potential HAMLET target. The binding between HAMLET and α-actinins was confirmed by co-immunoprecipitation and the actin and integrin binding sites on α-actinin were identified as possible sites of HAMLET binding in a peptide binding assay. HAMLET was also shown to colocalize with α-actinin-4 in the cell periphery and to reduce its granular surface staining and intracellular trabecular staining. In addition, HAMLET altered the staining of other focal adhesion components and reduced the level of active focal adhesion kinase and ERK, which can both be regulated via focal adhesions. Healthy cells did not detach after HAMLET treatment but the detachment of cancer cells was even further enhanced by knockdown of α-actinins. In contrast, overexpression of α-actinin-4-GFP delayed blebbing and rounding up further suggesting that α-actinin is involved in HAMLET-induced detachment. In summary, this study indicates that HAMLET activates autophagy and disrupts metabolism at least partly by binding to HK1 and that these events contribute to cell death. The results also suggest that c-Myc and glycolysis are important determinants of HAMLET sensitivity. Furthermore, the study identifies prohibitins as novel HAMLET targets with a potential role in cell death and suggests that HAMLET binding to α-actinins disrupts focal adhesions leading to cell detachment

Artificial intelligence : — a vehicle or an obstacle on our path to a sustainable future?

Many people and organizations across the globe work towards a sustainable future. Artificial intelligence (AI) can be a powerful tool in their efforts.AI technologies, which aim to imitate the natural intelligence of humans and animals, have made enormous progress in the past decade. AI has brought us a whole range of futuristic tools — from self-driving cars to virtual assistants, smart home appliances and self-learning robots — and enabled us to process large and complex data with unprecedented speed and accuracy. With AI advancing so rapidly and its application becoming more ubiquitous across society every day, we should carefully consider how to use it to ensure a sustainable future

Lysosomal cell death at a glance

Lysosomes serve as the cellular recycling centre and are filled with numerous hydrolases that can degrade most cellular macromolecules. Lysosomal membrane permeabilization and the consequent leakage of the lysosomal content into the cytosol leads to so-called "lysosomal cell death". This form of cell death is mainly carried out by the lysosomal cathepsin proteases and can have necrotic, apoptotic or apoptosis-like features depending on the extent of the leakage and the cellular context. This article summarizes our current knowledge on lysosomal cell death with an emphasis on the upstream mechanisms that lead to lysosomal membrane permeabilization

An annotated high-content fluorescence microscopy dataset with Hoechst 33342-stained nuclei and manually labelled outlines : Dataset record

Here we present a benchmarking dataset of fluorescence microscopy images with Hoechst 33342-stained nuclei together with annotations of nuclei, nuclear fragments and micronuclei. Images were randomly selected from an RNA interference screen with a modified U2OS osteosarcoma cell line, acquired on a Thermo Fischer CX7 high-content imaging system at 20x magnification. Labelling was performed by a single annotator and reviewed by a biomedical expert.The dataset contains 50 images showing over 2000 labelled nuclear objects in total, which is sufficiently large to train well-performing neural networks for instance or semantic segmentation. It is pre-split into training, development and test set, each in a zip file. The dataset should be referred to as Aitslab_bioimaging1. A preprint of a brief article describing the dataset is also available from zenodo (Arvidsson M, Kazemi Rashed S, Aits S. zenodo 2022, https://doi.org/10.1016/j.dib.2022.108769

An annotated high-content fluorescence microscopy dataset with Hoechst 33342-stained nuclei and manually labelled outlines : Dataset record

Here we present a benchmarking dataset of fluorescence microscopy images with Hoechst 33342-stained nuclei together with annotations of nuclei, nuclear fragments and micronuclei. Images were randomly selected from an RNA interference screen with a modified U2OS osteosarcoma cell line, acquired on a Thermo Fischer CX7 high-content imaging system at 20x magnification. Labelling was performed by a single annotator and reviewed by a biomedical expert.The dataset contains 50 images showing over 2000 labelled nuclear objects in total, which is sufficiently large to train well-performing neural networks for instance or semantic segmentation. It is pre-split into training, development and test set, each in a zip file. The dataset should be referred to as Aitslab_bioimaging1. A preprint of a brief article describing the dataset is also available from zenodo (Arvidsson M, Kazemi Rashed S, Aits S. zenodo 2022, https://doi.org/10.1016/j.dib.2022.108769