ROS release by PPARβ/δ-null fibroblasts reduces tumor load through epithelial antioxidant response.

Tumor stroma has an active role in the initiation, growth, and propagation of many tumor types by secreting growth factors and modulating redox status of the microenvironment. Although PPARβ/δ in fibroblasts was shown to modulate oxidative stress in the wound microenvironment, there has been no evidence of a similar effect in the tumor stroma. Here, we present evidence of oxidative stress modulation by intestinal stromal PPARβ/δ, using a FSPCre-Pparb/d <sup>-/-</sup> mouse model and validated it with immortalized cell lines. The FSPCre-Pparb/d <sup>-/-</sup> mice developed fewer intestinal polyps and survived longer when compared with Pparb/d <sup>fl/fl</sup> mice. The pre-treatment of FSPCre-Pparb/d <sup>-/-</sup> and Pparb/d <sup>fl/fl</sup> with antioxidant N-acetyl-cysteine prior DSS-induced tumorigenesis resulted in lower tumor load. Gene expression analyses implicated an altered oxidative stress processes. Indeed, the FSPCre-Pparb/d <sup>-/-</sup> intestinal tumors have reduced oxidative stress than Pparb/d <sup>fl/fl</sup> tumors. Similarly, the colorectal cancer cells and human colon epithelial cells also experienced lower oxidative stress when co-cultured with fibroblasts depleted of PPARβ/δ expression. Therefore, our results establish a role for fibroblast PPARβ/δ in epithelial-mesenchymal communication for ROS homeostasis

The role of fibroblast PPARβ/δ in skin homeostasis

The skin is the largest organ in the body and is made up of mainly the epidermis and dermis. The skin functions as a natural barrier against external stimuli, dehydration and injury. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is one member of the PPAR family of nuclear receptors, and the predominant subtype found in the epidermis and dermis. Epidermal PPARβ/δ has well-established roles in maintaining skin homeostasis, such as during wound healing. However, in the mammalian system, skin homeostasis depends on a complex crosstalk between the epithelial and mesenchymal compartments, underscoring the importance for a tight regulation of the complex epiethelial-mesenchymal interaction. Although epidermal PPARβ/δ in normal wound healing is well-studied, there has been no progress in clinical therapies for diabetic chronic wounds. This is also ensued with problems such as drug bioavailability and patients’ compliance. In our study, we showed that pharmacological activation of fibroblasts PPARβ/δ activity using microencapsulated ligands can promote the healing of diabetic wounds via the modulation of ROS production. The role of PPARβ/δ in other diseases, such as cancer, has also been controversial due to differences in experimental setups and genetic modifications of the PPARβ/δ gene. While epidermal PPARβ/δ has been widely investigated on in these diseases, the role of fibroblasts PPARβ/δ in skin homeostasis and disease has been neglected. Hence, we generated a novel mouse model with the PPARβ/δ gene deleted specifically in the fibroblasts. These animal exhibited skin abnormalities during development, such as dermal thickening and increased collagen production, recapitulating a morphea-like phenotype. In addition, the dermis manifest as a hotbed for inflammatory events. In our subsequent preliminary studies, we showed that cancer-associated fibroblasts from squamous cell carcinoma exhibit low levels of PPARβ/δ. Our novel mouse model will therefore serve as an appropriate model to study the role of fibroblasts PPARβ/δ in diseases, such as acute inflammation, wound healing and tumorigenesis in the future.DOCTOR OF PHILOSOPHY (SBS

Differential role of VEGF and ANGPTL4 in placental angiogenesis.

The placenta is crucial for nutrient exchange at the maternal-fetal interface. Placental angiogenesis is regulated by angiogenic factors like vascular endothelial growth factor(VEGF) and angiopoietin-like protein 4 (ANGPTL4) that are produced by trophoblast giant cells. However, the differential roles of VEGF and ANGPTL4 in placental angiogenesis remain unclear. Using trophoblast stem cells and mouse placenta, we investigated the different roles of VEGF and ANGPTL4 by establishing a spatiotemporal expression profile. By in vitro differentiation of trophoblast stem cells, our results showed that trophoblast cells produced VEGF and ANGPTL4, albeit displaying a different expression pattern. VEGF was readily detected during early differentiation, whereas ANGPTL4 was only detected much later. In contrast to VEGF, ANGPTL4 resulted in a prolonged disruption of endothelial tight junction integrity, as evidenced by the transendothelial electrical resistance assay and a redistribution of zona occludens-1 protein. These observations suggested that angiogenesis may require the priming of endothelial cells and augmentation of the angiogenic effect by VEGF and ANGPTL4, respectively. The reduced expression of VEGF in late-stage pregnancy permitted ANGPTL4 to adopt a more prominent role in late placental angiogenesis. The dysregulation of angiogenic factors expression will impair angiogenesis, threatens placental development and leads to miscarriages.Bachelor of Science in Biological Science

Emerging roles of angiopoietin-like 4 in human cancer

Angiopoietin-like 4 (ANGPTL4) is best known for its role as an adipokine involved in the regulation of lipid and glucose metabolism. The characterization of ANGPTL4 as an adipokine is largely due to our limited understanding of the interaction partners of ANGPTL4 and how ANGPTL4 initiates intracellular signaling. Recent findings have revealed a critical role for ANGPTL4 in cancer growth and progression, anoikis resistance, altered redox regulation, angiogenesis, and metastasis. Emerging evidence suggests that ANGPTL4 function may be drastically altered depending on the proteolytic processing and posttranslational modifications of ANGPTL4, which may clarify several conflicting roles of ANGPTL4 in different cancers. Although the N-terminal coiled-coil region of ANGPTL4 has been largely responsible for the endocrine regulatory role in lipid metabolism, insulin sensitivity, and glucose homeostasis, it has now emerged that the COOH-terminal fibrinogen-like domain of ANGPTL4 may be a key regulator in the multifaceted signaling during cancer development. New insights into the mechanistic action of this functional domain have opened a new chapter into the possible clinical application of ANGPTL4 as a promising candidate for clinical intervention in the fight against cancer. This review summarizes our current understanding of ANGPTL4 in cancer and highlights areas that warrant further investigation. A better understanding of the underlying cellular and molecular mechanisms of ANGPTL4 will reveal novel insights into other aspects of tumorigenesis and the potential therapeutic value of ANGPTL4.Accepted versio

Control Biosmart de sistemas de ventilación mediante recuperador de calor

El presente TFM está motivado por la creciente necesidad de controlar la calidad de aire interior (IAQ) de los edificios, donde las personas pasan actualmente más del 80% de su tiempo. La empresa inBiot, donde se ha desarrollado este trabajo, hace de ingeniería para temas relacionadas con esta temática. En este proyecto se ha mejorado la conectividad de los recuperadores de calor, permitiendo al usuario controlar el dispositivo desde la plataforma web de la empresa. Una vez validado este control, se procedió al desarrollo de un modo de trabajo en el que el recuperador de calor funcionara según la calidad del aire interior. Por otro lado, se ha diseñado un túnel de viento para poder caracterizar su funcionamiento y comprobar cómo se veían influenciados una serie de parámetros en el recuperador de calor.The present TFM is motivated by the growing need to control the indoor air quality (IAQ) in the buildings, where people currently spend more than 80% of time. The company inBiot, where this work has been developed, does engineering for topics related to the theme. In this project the connectivity of the heat recuperators has been improved, allowing the user being able to control the device from the company’s web platform. Once this control had been validated, a work mode was developed in order to the heat recovery unit works according to indoor air quality. By other side, a wind tunnel has been designed to characterize its operation and check out how series of heat recovery parameters where influenced.Máster Universitario en Ingeniería Industrial por la Universidad Pública de NavarraNafarroako Unibertsitate Publikoko Unibertsitate Masterra Industria Ingeniaritza

Probing for protein-protein interactions during cell migration: limitations and challenges

Cellular migration is a fundamental biological process occurring as early as embryogenesis to the pathological state of cancer metastasis. Nearly all cellular migrations involve an extracellular signal that is transduced internally by members of a signalling cascade. These signal transduction events are driven by protein-protein interactions (PPIs) that coordinate intracellular activities to enable a cell to migrate. Understanding these PPIs will provide valuable insight into how cellular migration can be modulated perhaps towards a therapeutic end. Histologically, not many techniques are available to demonstrate PPIs. Contrasting agents only demonstrate the presence of a particular protein, and perhaps its co-localisation with another protein. Yet, co-localisation need not necessarily indicate physical interaction between the two proteins. In this review, we highlight four commonly used methods that continue to expand our understanding of PPIs underlying cell migration: co-immunoprecipitation, bimolecular fluorescence complementation, proximity ligation assay and surface plasmon resonance. The methods discussed herein allow for the study of PPIs in a wide variety of biological samples, including cell lysates, live cells, fixed cells and tissues, enabling the quantification of endogenous PPIs and exploration of the nature of PPIs. We also include a rudimentary framework for researchers to decide which experimental method best suits their research goals

ROS release by PPARβ/δ-null fibroblasts reduces tumor load through epithelial antioxidant response

Tumor stroma has an active role in the initiation, growth, and propagation of many tumor types by secreting growth factors and modulating redox status of the microenvironment. Although PPARβ/δ in fibroblasts was shown to modulate oxidative stress in the wound microenvironment, there has been no evidence of a similar effect in the tumor stroma. Here, we present evidence of oxidative stress modulation by intestinal stromal PPARβ/δ, using a FSPCre-Pparb/d−/− mouse model and validated it with immortalized cell lines. The FSPCre-Pparb/d−/− mice developed fewer intestinal polyps and survived longer when compared with Pparb/dfl/fl mice. The pre-treatment of FSPCre-Pparb/d−/− and Pparb/dfl/fl with antioxidant Nacetyl- cysteine prior DSS-induced tumorigenesis resulted in lower tumor load. Gene expression analyses implicated an altered oxidative stress processes. Indeed, the FSPCre-Pparb/d−/− intestinal tumors have reduced oxidative stress than Pparb/dfl/fl tumors. Similarly, the colorectal cancer cells and human colon epithelial cells also experienced lower oxidative stress when co-cultured with fibroblasts depleted of PPARβ/δ expression. Therefore, our results establish a role for fibroblast PPARβ/δ in epithelial–mesenchymal communication for ROS homeostasis

Conditional knock out of N-WASP in keratinocytes causes skin barrier defects and atopic dermatitis-like inflammation

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously and regulates actin cytoskeleton remodeling. In order to characterize the role of N-WASP in epidermal homeostasis and cutaneous biology, we generated conditional N-WASP knockout mouse using CK14-cre (cytokeratin 14) to ablate expression of N-WASP in keratinocytes. N-WASPK14KO (N-WASP fl/fl ; CK14-Cre) mice were born following Mendelian genetics suggesting that N-WASP expression in keratinocytes is not essential during embryogenesis. N-WASPK14KO mice exhibited stunted growth, alopecia, dry and wrinkled skin. The dry skin in N-WASPK14KO mice is probably due to increased transepidermal water loss (TEWL) caused by barrier function defects as revealed by dye penetration assay. N-WASPK14KO mice developed spontaneous inflammation in the neck and face 10 weeks after birth. Histological staining revealed thickening of the epidermis, abnormal cornified layer and extensive infiltration of immune cells (mast cells, eosinophils and T-lymphocytes) in N-WASPK14KO mice skin compared to control mice. N-WASPK14KO mice had higher serum levels of IL-1α, TNF-α, IL-6 and IL-17 compared to control mice. Thus our results suggest that conditional N-WASP knockout in keratinocytes leads to compromised skin barrier, higher infiltration of immune cells and hyperproliferation of keratinocytes due to increased production of cytokines highlighting the importance of N-WASP in maintaining the skin homeostasis.MOE (Min. of Education, S’pore)Published versio

Targeting nuclear receptors in cancer-associated fibroblasts as concurrent therapy to inhibit development of chemoresistant tumors

Most anticancer therapies to date focus on druggable features of tumor epithelia. Despite the increasing repertoire of treatment options, patient responses remain varied. Moreover, tumor resistance and relapse remain persistent clinical challenges. These observations imply an incomplete understanding of tumor heterogeneity. The tumor microenvironment is a major determinant of disease progression and therapy outcome. Cancer-associated fibroblasts (CAFs) are the dominant cell type within the reactive stroma of tumors. They orchestrate paracrine pro-tumorigenic signaling with adjacent tumor cells, thus exacerbating the hallmarks of cancer and accelerating tumor malignancy. Although CAF-derived soluble factors have been investigated for tumor stroma-directed therapy, the underlying transcriptional programs that enable the oncogenic functions of CAFs remain poorly understood. Nuclear receptors (NRs), a large family of ligand-responsive transcription factors, are pharmacologically viable targets for the suppression of CAF-facilitated oncogenesis. In this study, we defined the expression profiles of NRs in CAFs from clinical cutaneous squamous cell carcinoma (SCC) biopsies. We further identified a cluster of driver NRs in CAFs as important modifiers of CAF function with profound influence on cancer cell invasiveness, proliferation, drug resistance, energy metabolism and oxidative stress status. Importantly, guided by the NR profile of CAFs, retinoic acid receptor β and androgen receptor antagonists were identified for concurrent therapy with cisplatin, resulting in the inhibition of chemoresistance in recurred SCC:CAF xenografts. Our work demonstrates that treatments targeting both the tumor epithelia and the surrounding CAFs can extend the efficacy of conventional chemotherapy.MOE (Min. of Education, S’pore)Published versio

Cancer-associated fibroblasts enact field cancerization by promoting extratumoral oxidative stress

Histological inspection of visually normal tissue adjacent to neoplastic lesions often reveals multiple foci of cellular abnormalities. This suggests the presence of a regional carcinogenic signal that spreads oncogenic transformation and field cancerization. We observed an abundance of mutagenic reactive oxygen species in the stroma of cryosectioned patient tumor biopsies, indicative of extratumoral oxidative stress. Diffusible hydrogen peroxide (H2O2) was elevated in the conditioned medium of cultured skin epithelia at various stages of oncogenic transformation, and H2O2 production increased with greater tumor-forming and metastatic capacity of the studied cell lines. Explanted cancer-associated fibroblasts (CAFs) also had higher levels of H2O2 secretion compared with normal fibroblasts (FIBs). These results suggest that extracellular H2O2 acts as a field effect carcinogen. Indeed, H2O2-treated keratinocytes displayed decreased phosphatase and tensin homolog (PTEN) and increased Src activities because of oxidative modification. Furthermore, treating FIBs with CAF-conditioned medium or exogenous H2O2 resulted in the acquisition of an oxidative, CAF-like state. In vivo, the proliferative potential and invasiveness of composite tumor xenografts comprising cancerous or non-tumor-forming epithelia with CAFs and FIBs could be attenuated by the presence of catalase. Importantly, we showed that oxidatively transformed FIBs isolated from composite tumor xenografts retained their ability to promote tumor growth and aggressiveness when adoptively transferred into new xenografts. Higher H2O2 production by CAFs was contingent on impaired TGFβ signaling leading to the suppression of the antioxidant enzyme glutathione peroxidase 1 (GPX1). Finally, we detected a reduction in Smad3, TAK1 and TGFβRII expression in a cohort of 197 clinical squamous cell carcinoma (SCC) CAFs, suggesting that impaired stromal TGFβ signaling may be a clinical feature of SCC. Our study indicated that CAFs and cancer cells engage redox signaling circuitries and mitogenic signaling to reinforce their reciprocal relationship, suggesting that future anticancer approaches should simultaneously target ligand receptor and redox-mediated pathways.MOE (Min. of Education, S’pore)Published versio