TGFβ signaling and the control of myofibroblast differentiation: Implications for chronic inflammatory disorders

The myofibroblast is a highly specialized cell type that plays a critical role during normal tissue wound healing, but also contributes pathologically to chronic inflammatory conditions such as fibrosis and cancer. As fibrotic conditions continue to be a major burden to the public health system, novel therapies that target the function of myofibroblasts may show promise in the clinic. The cytokine transforming growth factor β (TGFβ) is the most potent known inducer of myofibroblast differentiation and thus represents a powerful target to modify myofibroblast function during disease. This review focuses on our current understanding of the key signaling pathways activated by TGFβ during myofibroblast differentiation.     

Modulation of inflammation by autophagy: Consequences for human disease

Autophagy and inflammation are 2 fundamental biological processes involved in both physiological and pathological conditions. Through its crucial role in maintaining cellular homeostasis, autophagy is involved in modulation of cell metabolism, cell survival, and host defense. Defective autophagy is associated with pathological conditions such as cancer, autoimmune disease, neurodegenerative disease, and senescence. Inflammation represents a crucial line of defense against microorganisms and other pathogens, and there is increasing evidence that autophagy has important effects on the induction and modulation of the inflammatory reaction; understanding the balance between these 2 processes may point to important possibilities for therapeutic targeting. This review focuses on the crosstalk between autophagy and inflammation as an emerging field with major implications for understanding the host defense on the one hand, and for the pathogenesis and treatment of immune-mediated diseases on the other hand.     

ERK5: Structure, regulation and function

Extracellular signal-regulated kinase 5 (ERK5), also termed big mitogen-activated protein kinase-1 (BMK1), is the most recently identified member of the mitogen-activated protein kinase (MAPK) family and consists of an amino-terminal kinase domain, with a relatively large carboxy-terminal of unique structure and function that makes it distinct from other MAPK members. It is ubiquitously expressed in numerous tissues and is activated by a variety of extracellular stimuli, such as cellular stresses and growth factors, to regulate processes such as cell proliferation and differentiation. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade plays a critical role in cardiovascular development and vascular integrity. Recent data points to a potential role in pathological conditions such as cancer and tumour angiogenesis. This review focuses on the physiological and pathological role of ERK5, the regulation of this kinase and the recent development of small molecule inhibitors of the ERK5 signalling cascade.     

MiR-574–5p promotes the differentiation of human cardiac fibroblasts via regulating ARID3A

Cardiac fibrosis after myocardial infarction (MI) is mainly associated with cardiac fibroblasts and its differentiation is the key pathological process. However, the cellular mechanism of fibroblast-to-myofibroblast conversion has not been clarified and a deeper mechanistic understanding is needed. We found that miR-574–5p was up-regulated in TGF-β-induced myofibroblast differentiation. Silencing transiently miR-574–5p in HCFs, we found that suppression of miR-574–5p decreased myofibroblasts differentiation as validated by expression levels of fibrosis related genes, EDU imaging assay, wound healing assay and transwell assays. Conversely, overexpression of miR-574–5p displayed opposite results. ARID3A was verified as a direct target gene of miR-574–5p and decreased level of ARID3A forced fibroblast-to-myofibroblast differentiation of TGF-β-induced HCFs. Our data suggests that miR-574–5p plays a pivotal role in human cardiac fibroblasts (HCFs) myofibroblast differentiation and demonstrates that miR-574–5p and arid3a may be a novel therapeutic target for cardiac fibrosis.     

Computational analysis of the influence of the microenvironment on carcinogenesis

The tumour microenvironment is known to play an important role in determining the sequence of acquired phenotypic traits that characterise cancer evolution. A more precise understanding of this role could have a major influence in the understanding of cancer growth and development, and potentially in the optimisation of innovative anti-cancer treatments delivery. However, to lead such an analysis in the basis of traditional biological experiments and observations is almost utopian given the complexity of the underlying biological processes and the typical time scales involved. In this context, computer models constitute a complementary exploratory tool.In this paper we introduce a two-dimensional cellular automaton that models key cancer cell capabilities. The model has been especially designed to mimic the behaviour of a cancer colony growing in vitro and to analyse the effect of different environmental conditions on the sequence of acquisition of phenotypic traits. Our results indicate that microenvironmental factors such as the local concentration of oxygen or nutrients and cell overcrowding may determine the expansion of the tumour colony. The results also show that tumour cells evolve and that their phenotypes adapt to the microenvironment so that environmental stress determines the dominance of particular phenotypical traits.     

LIM-domain proteins in transforming growth factor β-induced epithelial-to-mesenchymal transition and myofibroblast differentiation

Epithelial to mesenchymal transition (EMT) is a process during which junctions of the cell-cell contacts are dissolved, actin cytoskeleton is deformed, apical-basolateral cell polarity is lost and cell motility is increased. EMT is needed during normal embryonal development and wound healing, but may also lead to pathogenic transformation and formation of myofibroblasts. Transforming growth factor β (TGFβ) is a multifunctional cytokine promoting EMT and myofibroblast differentiation, and its dysregulation is involved in pathological disorders like cancer and fibrosis. Lin11, Isl-1 and Mec-3 (LIM) domain proteins are associated with actin cytoskeleton and linked to regulation of cell growth, damage signaling, cell fate determination and signal transduction. LIM-domain proteins generally do not bind DNA, but are more likely to function via protein-protein interactions. Despite being a disparate group of proteins, similarities in their functions are observed. In this review we will discuss the role of LIM-domain proteins in TGFβ-signaling pathway and in EMT-driven processes. LIM-domain proteins regulate TGFβ-induced actin cytoskeleton reorganization, motility and adhesion, but also dissolution of cell-cell junctions during EMT. Finally, the role of LIM-domain proteins in myofibroblasts found in fibrotic foci and tumor stroma will be discussed.     

The role of Pdcd4 in tumour suppression and protein translation

Programmed cell death 4 (Pdcd4), a tumour suppressor, is frequently down‐regulated in various types of cancer. Pdcd4 has been demonstrated to efficiently suppress tumour promotion, progression and proliferation. The biochemical function of Pdcd4 is a protein translation inhibitor. Although the fact that Pdcd4 inhibits protein translation has been known for more than a decade, the mechanism by which Pdcd4 controls tumorigenesis through translational regulation of its target genes is still not fully understood. Recent studies show that Pdcd4 inhibits translation of stress‐activated‐protein kinase interacting protein 1 to suppress tumour invasion, depicting a picture of how Pdcd4 inhibits tumorigenesis through translational inhibition. Thus, understanding the mechanism of how Pdcd4 attenuates tumorigenesis by translational control should provide a new strategy for combating cancer.     

Protein tyrosine kinases: autoregulation and small-molecule inhibition

Receptor and non-receptor protein tyrosine kinases (PTKs) are essential enzymes in cellular signaling processes that regulate cell growth, differentiation, migration and metabolism. The kinase activity of PTKs is tightly controlled through steric, autoregulatory mechanisms, as well as by the action of protein tyrosine phosphatases. Recent structural studies have revealed several modes of autoregulation governing the catalytic state of these enzymes. Aberrant catalytic activity of many PTKs, via mutation or overexpression, plays an important role in numerous pathological conditions, including cancer. Structural studies of the Abl tyrosine kinase domain in complex with the small-molecule inhibitor STI571 provide a molecular basis for understanding the specificity determinants of this highly successful drug used in the treatment of chronic myeloid leukemia.     

Histidine-rich glycoprotein binds heparanase and regulates its enzymatic activity and cell surface interactions

Heparanase, an endo-β-d-glucuronidase, is involved in numerous normal physiological and pathological processes, such as inflammation, wound healing and tumour metastasis/angiogenesis, through its ability to mediate the degradation of heparan sulfate, a key structural component of the extracellular matrix and on the surface of cells. Identifying endogenous molecules that can regulate heparanase activity will aid the understanding of its molecular function in health and disease and provide the potential for development of novel anti-cancer and anti-inflammatory therapeutics. The ability of the extracellular heparanase to tether onto cell surface heparan sulfate proteoglycans and other receptor(s), such as the cation-independent mannose-6-phosphate receptor, is key to its activation, function and uptake into intracellular compartments. Here we describe experiments demonstrating that a relatively abundant plasma glycoprotein, histidine-rich glycoprotein, directly interacts with platelet-derived heparanase and enhances its enzymatic activity. The findings in this study also show that histidine-rich glycoprotein interferes with heparanase binding to cell surface receptors, particularly heparan sulfate proteoglycans. Thus, the interaction between histidine-rich glycoprotein and heparanase can potentially regulate the role of heparanase in a variety of physiological and pathological conditions.     

The cancerous translation apparatus

Deregulations in translational control are critical features of cancer initiation and progression. Activation of key oncogenic pathways promotes rapid and dramatic translational reprogramming, not simply by increasing overall protein synthesis, but also by modulating specific mRNA networks that promote cellular transformation. Additionally, ribosomopathies caused by mutations in ribosome components alter translational regulation leading to specific pathological features, including cancer susceptibility. Exciting advances in our understanding of translational control in cancer have illuminated a striking specificity innate to the translational apparatus. Characterizing this specificity will provide novel insights into how cells normally utilize translational control to modulate gene expression, how it is deregulated in cancer, and how these processes can be targeted to develop new cancer therapies.     

Cancer research by means of tissue engineering – is there a rationale?

Tissue engineering (TE) has evoked new hopes for the cure of organ failure and tissue loss by creating functional substitutes in the laboratory. Besides various innovations in the context of Regenerative Medicine (RM), TE also provided new technology platforms to study mechanisms of angiogenesis and tumour cell growth as well as potentially tumour cell spreading in cancer research. Recent advances in stem cell technology – including embryonic and adult stem cells and induced pluripotent stem cells – clearly show the need to better understand all relevant mechanisms to control cell growth when such techniques will be administered to patients. Such TE‐Cancer research models allow us to investigate the interactions that occur when replicating physiological and pathological conditions during the initial phases of replication, morphogenesis, differentiation and growth under variable given conditions. Tissue microenvironment has been extensively studied in many areas of TE and it plays a crucial role in cell signalling and regulation of normal and malignant cell functions. This article is intended to give an overview on some of the most recent developments and possible applications of TE and RM methods with regard to the improvement of cancer research with TE platforms. The synthesis of TE with innovative methods of molecular biology and stem‐cell technology may help investigate and potentially modulate principal phenomena of tumour growth and spreading, as well as tumour‐related angiogenesis. In the future, these models have the potential to investigate the optimal materials, culture conditions and material structure to propagate tumour growth.     

Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Small cell lung cancer (SCLC) accounts for nearly 15% of human lung cancers and is one of the most aggressive solid tumors. The SCLC cells are thought to derive from self-renewing pulmonary neuroendocrine cells by oncogenic transformation. However, whether the SCLC cells possess stemness and plasticity for differentiation as normal stem cells has not been well understood thus far. In this study, we investigated the expressions of multilineage stem cell markers in the cancer cells of SCLC cell line (NCI-H446) and analyzed their clonogenicity, tumorigenicity, and plasticity for inducing differentiation. It has been found that most cancer cells of the cell line expressed multilineage stem cell markers under the routine culture conditions and generated single-cell clones in anchorage-dependent or -independent conditions. These cancer cells could form subcutaneous xenograft tumors and orthotopic lung xenograft tumors in BALB/C-nude mice. Most cells in xenograft tumors expressed stem cell markers and proliferation cell nuclear antigen Ki67, suggesting that these cancer cells remained stemness and highly proliferative ability in vivo. Intriguingly, the cancer cells could be induced to differentiate into neurons, adipocytes, and osteocytes, respectively, in vitro. During the processes of cellular phenotype-conversions, autophagy and apoptosis were two main metabolic events. There is cross-talking between autophagy and apoptosis in the differentiated cancer cells. In addition, the effects of the inhibitor and agonist for Sirtuin1/2 on the inducing osteogenic differentiation indicated that Sirtuin1/2 had an important role in this process. Taken together, these results indicate that most cancer cells of NCI-H446 cell line possess stemness and plasticity for multilineage differentiation. These findings have potentially some translational applications in treatments of SCLC with inducing differentiation therapy.     

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle -actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.     

Molecular mechanisms and physiological functions of mitophagy

Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy‐mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady‐state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations.     

Brca1 and differentiation

Breast cancer is one of the most frequent malignancies affecting women. The human breast cancer gene 1 (BRCA1) gene is mutated in a distinct proportion of hereditary breast and ovarian cancers. Tumourigenesis in individuals with germline BRCA1 mutations requires somatic inactivation of the remaining wild-type allelle. Although, this evidence supports a role for BRCA1 as a tumour suppressor, the mechanisms through which its loss leads to tumourigenesis remain to be determined. Neither the expression pattern nor the described functions of human BRCA1 and murine breast cancer gene 1 (Brca1) can explain the specific association of mutations in this gene with the development of breast and ovarian cancer. Investigation of the role of Brca1 in normal cell differentiation processes might provide the basis to understand the tissue-restricted properties.     

Unc119 regulates myofibroblast differentiation through the activation of Fyn and the p38 MAPK pathway

Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of alpha-smooth muscle actin (alpha-SMA) and myofibroblast differentiation by TGF-beta in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-beta and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate alpha-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of alpha-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced alpha-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-beta and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of alpha-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.