Recent advances on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology

Cholangiocytes are epithelial cells lining the biliary epithelium. Cholangiocytes play several key roles in the modification of ductal bile and are also the target cells in chronic cholestatic liver diseases (i.e., cholangiopathies) such as PSC, PBC, polycystic liver disease (PCLD) and cholangiocarcinoma (CCA). During these pathologies, cholangiocytes (which in normal condition are in a quiescent state) begin to proliferate acquiring phenotypes of neuroendocrine cells, and start secreting different cytokines, growth factors, neuropeptides, and hormones to modulate cholangiocytes proliferation and interaction with the surrounding environment, trying to reestablish the balance between proliferation/loss of cholangiocytes for the maintenance of biliary homeostasis. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte pathophysiology. To clarify the mechanisms of action of these factors we will provide new potential strategies for the management of chronic liver diseases

The role of compartmentalized signaling pathways in the control of mitochondrial activities in cancer cells

Mitochondria are the powerhouse organelles present in all eukaryotic cells. They play a fundamental role in cell respiration, survival and metabolism. Stimulation of G-protein coupled receptors (GPCRs) by dedicated ligands and consequent activation of the cAMP·PKA pathway finely couple energy production and metabolism to cell growth and survival. Compartmentalization of PKA signaling at mitochondria by A-Kinase Anchor Proteins (AKAPs) ensures efficient transduction of signals generated at the cell membrane to the organelles, controlling important aspects of mitochondrial biology. Emerging evidence implicates mitochondria as essential bioenergetic elements of cancer cells that promote and support tumor growth and metastasis. In this context, mitochondria provide the building blocks for cellular organelles, cytoskeleton and membranes, and supply all the metabolic needs for the expansion and dissemination of actively replicating cancer cells. Functional interference with mitochondrial activity deeply impacts on cancer cell survival and proliferation. Therefore, mitochondria represent valuable targets of novel therapeutic approaches for the treatment of cancer patients. Understanding the biology of mitochondria, uncovering the molecular mechanisms regulating mitochondrial activity andmapping the relevant metabolic and signaling networks operating in cancer cells will undoubtly contribute to create a molecular platform to be used for the treatment of proliferative disorders. Here, we will highlight the emerging roles of signaling pathways acting downstream to GPCRs and their intersection with the ubiquitin proteasome system in the control of mitochondrial activity in different aspects of cancer cell biology

The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferatio

Defining the pro-tumour impact of the evolving stromal microenvironment

Much like normal tissues, tumours require a supporting microenvironment for growth and survival, known as the tumour stroma. However, tumours represent a dynamic and turbulent environment, in which factors such as hypoxia, fibrosis, nutrient deprivation and the local cytokine milieu continually fluctuate as the tumour grows and develops. These factors influence stromal phenotypes and create heterogeneity, which can confound our understanding of their role within the microenvironment. In particular, cancer associated fibroblasts (CAFs) represent a diverse population of cells, which cannot be identified by one universal marker. CAFs promote tumour growth and dissemination by secreting growth factors, stimulating angiogenesis, aiding the development of tumour-promoting inflammation and remodelling the extra-cellular matrix (ECM). However, recent investigations have shown these functions belong to defined populations that differ between tumour types. This project aimed to investigate stromal heterogeneity across melanoma development, with a specific focus on the CAF compartment. Whilst conventional techniques such as IF and flow cytometry showed varied expression of fibroblast markers, they lacked the resolution to discern functional subsets. Thus, we employed single cell RNA sequencing (scRNAseq) to profile CAF populations at different stages of tumour development. To avoid bias, CAFs were isolated from the B16-F10 melanoma model using a ‘negative selection’ approach. Our data revealed the presence of 3 functionally distinct fibroblast populations, termed ‘immune’, ‘desmoplastic’ and ‘contractile’, which expressed genes involved in immune cross talk, matrix remodelling and stress fibre contraction respectively. Furthermore, these populations are dynamic, changing in prevalence as the tumour grows. While ‘immune’ and ‘desmoplastic’ populations were present from early stages, ‘contractile’ CAFs were more abundant at later time points. Owing to their unique marker profiles, we were able to identify these populations within the tumour stoma and validate their temporal nature. Subsequent investigation into the contribution of these subsets to tumour growth, revealed that ‘immune’ CAFs promoted accumulation of suppressive macrophages by production of C3 and its cleavage product C3a. Significantly, inhibition of the C3a/C3aR axis reduced the number of macrophages and decreased tumour volume. This reduction in tumour growth was accompanied by increased CD8 T-cell infiltration, implying that ‘immune’ CAFs may inhibit adaptive anti-tumour immunity through controlling the myeloid compartment. The interaction between C3 producing CAFs and C3aR expressing macrophages was conserved in different murine tumours and human cancer. Thus, ‘immune’ CAFs and C3a signalling may represent therapeutic targets in multiple cancer types. Overall, our data highlights the complexity of stromal phenotypes and microenvironment interactions, which likely reflects the convoluted climate of the developing tumour

Absence of MHC-II expression by lymph node stromal cells results in autoimmunity.

How lymph node stromal cells (LNSCs) shape peripheral T-cell responses remains unclear. We have previously demonstrated that murine LNSCs, lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and fibroblastic reticular cells (FRCs) use the IFN-γ-inducible promoter IV (pIV) of the MHC class II (MHCII) transactivator CIITA to express MHCII. Here, we show that aging mice (>1 yr old) in which MHCII is abrogated in LNSCs by the selective deletion of pIV exhibit a significant T-cell dysregulation in LNs, including defective Treg and increased effector CD4 <sup>+</sup> and CD8 <sup>+</sup> T-cell frequencies, resulting in enhanced peripheral organ T-cell infiltration and autoantibody production. The proliferation of LN-Tregs interacting with LECs increases following MHCII up-regulation by LECs upon aging or after exposure to IFN-γ, this effect being abolished in mice in which LECs lack MHCII. Overall, our work underpins the importance of LNSCs, particularly LECs, in supporting Tregs and T-cell tolerance