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Cancers are often associated with hypoxia and metabolic reprogramming, resulting in enhanced tumor progression. Here, we aim to study breast cancer hypoxia responses, focusing on secreted proteins from low-grade (luminal-like) and high-grade (basal-like) cell lines before and after hypoxia. We examine the overlap between proteomics data from secretome analysis and laser microdissected human breast cancer stroma, and we identify a 33-protein stromal-based hypoxia profile (33P) capturing differences between luminal-like and basal-like tumors. The 33P signature is associated with metabolic differences and other adaptations following hypoxia. We observe that mRNA values for 33P predict patient survival independently of molecular subtypes and basic prognostic factors, also among low-grade luminal-like tumors. We find a significant prognostic interaction between 33P and radiation therapy. The role of hypoxia and metabolic reprogramming in breast cancer remains to be explored. Here, the authors investigate the landscape of secreted proteins in response to hypoxia in breast cancer cell lines and identify a stromal-based hypoxia profile in breast cancer tissue. [ABSTRACT FROM AUTHOR]

Background/Aim: Better stratification of the risk of relapse will help select the right patients for adjuvant treatment and improve targeted therapies for patients with colon cancer. Materials and Methods: To understand why a subset of tumors relapse, we compared the proteome of two groups of patients with colon cancer with similar stage, stratified based on the presence or absence of recurrence. Results: Using tumor biopsies from the primary operation, we identified dissimilarity between recurrent and nonrecurrent mismatch satellite stable colon cancer and found that signaling related to immune activation and inflammation was associated with relapse. Conclusion: Immune modulation may have an effect on mismatch satellite stable colon cancer. At present, immune therapy is offered primarily to microsatellite instable colon cancer. Hopefully, immune therapy in mismatch satellite stable colon cancer beyond PD-1 and PD-L1 inhibitors can be implemented. [ABSTRACT FROM AUTHOR]

Aim: The loss of insulin‐secreting β‐cells, ultimately characterizing most diabetes forms, demands the development of cell replacement therapies. The common endpoint for all ex vivo strategies is transplantation into diabetic patients. However, the effects of hyperglycaemia environment on the transplanted cells were not yet properly assessed. Thus, the main goal of this study was to characterize global effect of brief and prolonged in vivo hyperglycaemia exposure on the cell fate acquisition and maintenance of transplanted human pancreatic progenitors. Methods: To rigorously study the effect of hyperglycaemia, in vitro differentiated human‐induced pluripotent stem cells (hiPSC)‐derived pancreatic progenitors were xenotransplanted in normoglycaemic and diabetic NSG rat insulin promoter (RIP)‐diphtheria toxin receptor (DTR) mice. The transplants were retrieved after 1‐week or 1‐month exposure to overt hyperglycaemia and analysed by large‐scale microscopy or global proteomics. For this study we pioneer the use of the NSG RIP‐DTR system in the transplantation of hiPSC, making use of its highly reproducible specific and absolute β‐cell ablation property in the absence of inflammation or other organ toxicity. Results: Here we show for the first time that besides the presence of an induced oxidative stress signature, the cell fate and proteome landscape response to hyperglycaemia was different, involving largely different mechanisms, according to the period spent in the hyperglycaemic environment. Surprisingly, brief hyperglycaemia exposure increased the bihormonal cell number by impeding the activity of specific islet lineage determinants. Moreover, it activated antioxidant and inflammation protection mechanisms signatures in the transplanted cells. In contrast, the prolonged exposure was characterized by decreased numbers of hormone + cells, low/absent detoxification signature, augmented production of oxygen reactive species and increased apoptosis. Conclusion: Hyperglycaemia exposure induced distinct, period‐dependent, negative effects on xenotransplanted human pancreatic progenitor, affecting their energy homeostasis, cell fate acquisition and survival. [ABSTRACT FROM AUTHOR]

A comprehensive characterization of the molecular processes controlling cell fate decisions is essential to derive stable progenitors and terminally differentiated cells that are functional from human pluripotent stem cells (hPSCs). Here, we report the use of quantitative proteomics to describe early proteome adaptations during hPSC differentiation toward pancreatic progenitors. We report that the use of unbiased quantitative proteomics allows the simultaneous profiling of numerous proteins at multiple time points, and is a valuable tool to guide the discovery of signaling events and molecular signatures underlying cellular differentiation. We also monitored the activity level of pathways whose roles are pivotal in the early pancreas differentiation, including the Hippo signaling pathway. The quantitative proteomics data set provides insights into the dynamics of the global proteome during the transition of hPSCs from a pluripotent state toward pancreatic differentiation. [ABSTRACT FROM AUTHOR]

Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalling cascades promoting differentiation. These analyses provide a molecular framework for understanding the confinement effects on hiPSCs differentiation while confirming its importance for this process. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity. [ABSTRACT FROM AUTHOR]

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation. [ABSTRACT FROM AUTHOR]

Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting β-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-β-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases. Islet non-β-cells from non-diabetic and diabetic human donors are modified via the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. [ABSTRACT FROM AUTHOR]

Patients with bladder cancer need frequent controls over long follow-up time due to high recurrence rate and risk of conversion to muscle invasive cancer with poor prognosis. We identified cancer-related molecular signatures in apparently healthy bladder in patients with subsequent muscular invasiveness during follow-up. Global proteomics of the normal tissue biopsies revealed specific proteome fingerprints in these patients prior to subsequent muscular invasiveness. In these presumed normal samples, we detected modulations of proteins previously associated with different cancer types. This study indicates that analyzing apparently healthy tissue of a cancer-invaded organ may suggest disease progression. [ABSTRACT FROM AUTHOR]

Background It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. Methods In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography–tandem mass spectrometry. Results 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Conclusions Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats. [ABSTRACT FROM AUTHOR]

Abnormal hepatic insulin signaling is a cause or consequence of hepatic steatosis. DPP-4 inhibitors might be protective against fatty liver. We previously reported that the systemic inhibition of insulin receptor (IR) and IGF-1 receptor (IGF1R) by the administration of OSI-906 (linsitinib), a dual IR/IGF1R inhibitor, induced glucose intolerance, hepatic steatosis, and lipoatrophy in mice. In the present study, we investigated the effects of a DPP-4 inhibitor, linagliptin, on hepatic steatosis in OSI-906-treated mice. Unlike high-fat diet-induced hepatic steatosis, OSI-906-induced hepatic steatosis is not characterized by elevations in inflammatory responses or oxidative stress levels. Linagliptin improved OSI-906-induced hepatic steatosis via an insulin-signaling-independent pathway, without altering glucose levels, free fatty acid levels, gluconeogenic gene expressions in the liver, or visceral fat atrophy. Hepatic quantitative proteomic and phosphoproteomic analyses revealed that perilipin-2 (PLIN2), major urinary protein 20 (MUP20), cytochrome P450 2b10 (CYP2B10), and nicotinamide N-methyltransferase (NNMT) are possibly involved in the process of the amelioration of hepatic steatosis by linagliptin. Thus, linagliptin improved hepatic steatosis induced by IR and IGF1R inhibition via a previously unknown mechanism that did not involve gluconeogenesis, lipogenesis, or inflammation, suggesting the non-canonical actions of DPP-4 inhibitors in the treatment of hepatic steatosis under insulin-resistant conditions. [ABSTRACT FROM AUTHOR]

Mutations in the hepatocyte nuclear factor 4α (HNF4α) gene affect prenatal and postnatal pancreas development, being characterized by insulin-producing β-cell dysfunction. Little is known about the cellular and molecular mechanisms leading to β-cell failure as result of HNF4α mutation. In this study, we compared the miRNA profile of differentiating human induced pluripotent stem cells (hiPSC) derived from HNF4α+/Δ mutation carriers and their family control along the differentiation timeline. Moreover, we associated this regulation with the corresponding transcriptome profile to isolate transcript–miRNA partners deregulated in the mutated cells. This study uncovered a steep difference in the miRNA regulation pattern occurring during the posterior foregut to pancreatic endoderm transition, defining early and late differentiation regulatory windows. The pathway analysis of the miRNAome–transcriptome interactions revealed a likely gradual involvement of HNF4α+/Δ mutation in p53-mediated cell cycle arrest, with consequences for the proliferation potential, survival and cell fate acquisition of the differentiating cells. The present study is based on bioinformatics approaches and we expect that, pending further experimental validation, certain miRNAs deregulated in the HNF4α+/Δ cells would prove useful for therapy. [ABSTRACT FROM AUTHOR]