THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog.

Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insights into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine- or puromycin analogs allow for the selective visualization and enrichment of newly synthesized proteins. However, their applications are limited as they often require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a threonine-derived non-canonical amino acid tagging method based on the bioorthogonal threonine analog ß-ethynylserine (ßES) that enables efficient labeling of the nascent proteome in complete growth media within minutes. We use THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profile immediate proteome dynamics of B-cells in response to B-cell receptor activation simply by adding ßES to the culture medium, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in specific cell types in vivo.

TGF-ß Type I Receptor Signaling in Melanoma Liver Metastases Increases Metastatic Outgrowth.

Despite advances in treatment for metastatic melanoma patients, patients with liver metastasis have an unfavorable prognosis. A better understanding of the development of liver metastasis is needed. The multifunctional cytokine Transforming Growth Factor ß (TGF-ß) plays various roles in melanoma tumors and metastasis, affecting both tumor cells and cells from the surrounding tumor microenvironment. To study the role of TGF-ß in melanoma liver metastasis, we created a model to activate or repress the TGF-ß receptor pathway in vitro and in vivo in an inducible manner. For this, we engineered B16F10 melanoma cells to have inducible ectopic expression of a constitutively active (ca) or kinase-inactive (ki) TGF-ß receptor I, also termed activin receptor-like kinase (ALK5). In vitro, stimulation with TGF-ß signaling and ectopic caALK5 expression reduced B16F10 cell proliferation and migration. Contrasting results were found in vivo; sustained caALK5 expression in B16F10 cells in vivo increased the metastatic outgrowth in liver. Blocking microenvironmental TGF-ß did not affect metastatic liver outgrowth of both control and caALK5 expressing B16F10 cells. Upon characterizing the tumor microenvironment of control and caALk5 expressing B16F10 tumors, we observed reduced (cytotoxic) T cell presence and infiltration, as well as an increase in bone marrow-derived macrophages in caALK5 expressing B16F10 tumors. This suggests that caALK5 expression in B16F10 cells induces changes in the tumor microenvironment. A comparison of newly synthesized secreted proteins upon caALK5 expression by B16F10 cells revealed increased secretion of matrix remodeling proteins. Our results show that TGF-ß receptor activation in B16F10 melanoma cells can increase metastatic outgrowth in liver in vivo, possibly through remodeling of the tumor microenvironment leading to altered infiltration of immune cells. These results provide insights in the role of TGF-ß signaling in B16F10 liver metastasis and could have implications regarding the use of TGF-ß inhibitors for the treatment of melanoma patients with liver metastasis.

Multiomics of Colorectal Cancer Organoids Reveals Putative Mediators of Cancer Progression Resulting from SMAD4 Inactivation.

The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Although loss of SMAD4 is a key event in CRC progression, the resulting changes in biological processes in advanced disease and metastasis are not fully understood. Here, we applied a multiomics approach to a CRC organoid model that faithfully reflects the metastasis-supporting effects of SMAD4 inactivation. We show that loss of SMAD4 results in decreased differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFß, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by SMAD4-mutant organoids─DKK3─reduces the antitumor effects of natural killer cells (NK cells). Altogether, our data provide new insights into the role of SMAD4 perturbation in advanced CRC.

Human scattered tubular cells represent a heterogeneous population of glycolytic dedifferentiated proximal tubule cells.

Scattered tubular cells (STCs) are a phenotypically distinct cell population in the proximal tubule that increase in number after acute kidney injury. We aimed to characterize the human STC population. Three-dimensional human tissue analysis revealed that STCs are preferentially located within inner bends of the tubule and are barely present in young kidney tissue (<2 years), and their number increases with age. Increased STC numbers were associated with acute tubular injury (kidney injury molecule 1) and interstitial fibrosis (alpha smooth muscle actin). Isolated CD13+ CD24- CD133- proximal tubule epithelial cells (PTECs) and CD13+ CD24+ and CD13+ CD133+ STCs were analyzed using RNA sequencing. Transcriptome analysis revealed an upregulation of nuclear factor κB, tumor necrosis factor alpha, and inflammatory pathways in STCs, whereas metabolism, especially the tricarboxylic acid cycle and oxidative phosphorylation, was downregulated, without showing signs of cellular senescence. Using immunostaining and a publicly available single-cell sequencing database of human kidneys, we demonstrate that STCs represent a heterogeneous population in a transient state. In conclusion, STCs are dedifferentiated PTECs showing a metabolic shift toward glycolysis, which could facilitate cellular survival after kidney injury. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Retinoic acid signaling drives differentiation toward the absorptive lineage in colorectal cancer.

Retinoic acid (RA) signaling is an important and conserved pathway that regulates cellular proliferation and differentiation. Furthermore, perturbed RA signaling is implicated in cancer initiation and progression. However, the mechanisms by which RA signaling contributes to homeostasis, malignant transformation, and disease progression in the intestine remain incompletely understood. Here, we report, in agreement with previous findings, that activation of the Retinoic Acid Receptor and the Retinoid X Receptor results in enhanced transcription of enterocyte-specific genes in mouse small intestinal organoids. Conversely, inhibition of this pathway results in reduced expression of genes associated with the absorptive lineage. Strikingly, this latter effect is conserved in a human organoid model for colorectal cancer (CRC) progression. We further show that RXR motif accessibility depends on progression state of CRC organoids. Finally, we show that reduced RXR target gene expression correlates with worse CRC prognosis, implying RA signaling as a putative therapeutic target in CRC.

Androgen receptor signalling confers clonogenic and migratory advantages in urothelial cell carcinoma of the bladder.

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR-positive UCC-derived cell line model, UM-UC-3-AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell-like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen-stimulated UM-UC-3-AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.

Effect of dimethyl disulfide on the sulfur formation and microbial community composition during the biological H2S removal from sour gas streams.

Removal of organic and inorganic sulfur compounds from sour gases is required because of their toxicity and atmospheric pollution. The most common are hydrogen sulfide (H2S) and methanethiol (MT). Under oxygen-limiting conditions about 92 mol% of sulfide is oxidized to sulfur by haloalkaliphilic sulfur-oxidizing bacteria (SOB), whilst the remainder is oxidized either biologically to sulfate or chemically to thiosulfate. MT is spontaneously oxidized to dimethyl disulfide (DMDS), which was found to inhibit the oxidation of sulfide to sulfate. Hence, we assessed the effect of DMDS on product formation in a lab-scale biodesulfurization setup. DMDS was quantified using a newly, in-house developed analytical method. Subsequently, a chemical reaction mechanism was proposed for the formation of methanethiol and dimethyl trisulfide from the reaction between sulfide and DMDS. Addition of DMDS resulted in significant inhibition of sulfate formation, leading to 96 mol% of sulfur formation. In addition, a reduction in the dominating haloalkaliphilic SOB species, Thioalkalivibrio sulfidiphilus, was observed in favor of Thioalkaibacter halophilus as a more DMDS-tolerant with the 50 % inhibition coefficient at 2.37 mM DMDS.