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The incidence of nonmelanoma skin cancer (NMSC) has been increasing worldwide. Most studies have highlighted the importance of cancer-associated fibroblasts (CAFs) in NMSC progression. However much less is known about the communication between normal fibroblasts and epithelia; disruption of this communication affects tumor initiation and the latency period in the emergence of tumors. Delineating the mechanism that mediates this epithelial-mesenchymal communication in NMSC could identify more effective targeted therapies. The nuclear receptor PPARβ/δ in fibroblasts has been shown to modulate adjacent epithelial cell behavior, however, its role in skin tumorigenesis remains unknown. Using chemically induced skin carcinogenesis, we showed that FSPCre-Pparb/dex4 mice, whose Pparb/d gene was selectively deleted in fibroblasts, had delayed emergence and reduced tumor burden compared with control mice (Pparb/dfl/fl). However, FSPCre-Pparb/dex4-derived tumors showed increased proliferation, with no difference in differentiation, suggesting delayed tumor initiation. Network analysis revealed a link between dermal Pparb/d and TGF-β1 with epidermal NRF2 and Nox4. In vitro investigations showed that PPARβ/δ deficiency in fibroblasts increased epidermal Nox4-derived H2O2 production, which triggered an NRF2-mediated antioxidant response. We further showed that H2O2 upregulated NRF2 mRNA via the B-Raf-MEK1/2 pathway. The enhanced NRF2 response altered the activities of PTEN, Src, and AKT. In vivo, we detected the differential phosphorylation profiles of B-Raf, MEK1/2, PTEN, Src, and AKT in the vehicle-treated and chemically treated epidermis of FSPCre-Pparb/dex4 mice compared with that in Pparb/dfl/fl mice, prior to the first appearance of tumors in Pparb/dfl/fl. Our study revealed a role for fibroblast PPARβ/δ in the epithelial-mesenchymal communication involved in cellular redox homeostasis. Ministry of Education (MOE) Accepted version This research/project is supported by Start-Up Grant (M4082040) and Ministry of Education, Singapore, under Academic Research Fund Tier 1 (2017-T1-002-103) to NST, (2015-T1-001-034) to WW and Start-Up Grant from the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore to WW and XW; the Région Midi-Pyrénées through the Chaire d’Excellence Pierre de Fermat and the Bonizzi-Theler-Stiftung to WW; SERB-DST, Govt. of India funded Ramanujan Fellowship Grant (SB/S2/RJN-087/2014) to MP

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/d fl/fl mice. The pre-treatment of FSPCre-Pparb/d -/- and Pparb/d fl/fl 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 -/- intestinal tumors have reduced oxidative stress than Pparb/d fl/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.

Connective tissue diseases of the skin are characterized by excessive collagen deposition in the skin and internal organs. Fibroblasts play a pivotal role in the clinical presentation of these conditions. Nuclear receptor peroxisome-proliferator activated receptors (PPARs) are therapeutic targets for dermal fibrosis, but the contribution of the different PPAR subtypes are poorly understood. Particularly, the role of fibroblast PPARβ/δ in dermal fibrosis has not been elucidated. Thus, we generated a mouse strain with selective deletion of PPARβ/δ in the fibroblast (FSPCre-Pparb/d−/−) and interrogated its epidermal and dermal transcriptome profiles. We uncovered a downregulated gene, leucine-rich alpha-2-glycoprotein-1 (Lrg1), of previously unknown function in skin development and architecture. Our findings suggest that the regulation of Lrg1 by PPARβ/δ in fibroblasts is an important signaling conduit integrating PPARβ/δ and TGFβ1-signaling networks in skin health and disease. Thus, the FSPCre-Pparb/d−/− mouse model could serve as a novel tool in the current gunnery of animal models to better understand dermal fibrosis.

Many gastrointestinal diseases exhibit a protracted and aggravated inflammatory response that can lead to hypercytokinaemia, culminating in extensive tissue damage. Recently, angiopoietin-like 4 (ANGPTL4) has been implicated in many inflammation-associated diseases. However, how ANGPTL4 regulates colonic inflammation remains unclear. Herein, we show that ANGPTL4 deficiency in mice (ANGPTL4−/−) exacerbated colonic inflammation induced by dextran sulfate sodium (DSS) or stearic acid. Microbiota was similar between the two genotypes prior DSS challenge. A microarray gene expression profile of the colon from DSS-treated ANGPTL4−/− mice was enriched for genes involved in leukocyte migration and infiltration, and showed a close association to inflamed ulcerative colitis (UC), whereas the profile from ANGPTL4+/+ littermates resembled that of non-inflamed UC biopsies. Bone marrow transplantation demonstrates the intrinsic role of colonic ANGPTL4 in regulating leukocyte infiltration during DSS-induced inflammation. Using immortalized human colon epithelial cells, we revealed that the ANGPTL4-mediated upregulation of tristetraprolin expression operates through CREB and NF-κB transcription factors, which in turn, regulates the stability of chemokines. Together, our findings suggest that ANGPTL4 protects against acute colonic inflammation and that its absence exacerbates the severity of inflammation. Our findings emphasize the importance of ANGPTL4 as a novel target for therapy in regulating and attenuating inflammation.

Metastatic cancer cells acquire energy-intensive processes including increased invasiveness and chemoresistance. However, how the energy demand is met and the molecular drivers that coordinate an increase in cellular metabolic activity to drive epithelial–mesenchymal transition (EMT), the first step of metastasis, remain unclear. Using different in vitro and in vivo EMT models with clinical patient’s samples, we showed that EMT is an energy-demanding process fueled by glucose metabolism-derived adenosine triphosphate (ATP). We identified angiopoietin-like 4 (ANGPTL4) as a key player that coordinates an increase in cellular energy flux crucial for EMT via an ANGPTL4/14-3-3γ signaling axis. This augmented cellular metabolic activity enhanced metastasis. ANGPTL4 knockdown suppresses an adenylate energy charge elevation, delaying EMT. Using an in vivo dual-inducible EMT model, we found that ANGPTL4 deficiency reduces cancer metastasis to the lung and liver. Unbiased kinase inhibitor screens and Ingenuity Pathway Analysis revealed that ANGPTL4 regulates the expression of 14-3-3γ adaptor protein via the phosphatidylinositol-3-kinase/AKT and mitogen-activated protein kinase signaling pathways that culminate to activation of transcription factors, CREB, cFOS and STAT3. Using a different mode of action, as compared with protein kinases, the ANGPTL4/14-3-3γ signaling axis consolidated cellular bioenergetics and stabilized critical EMT proteins to coordinate energy demand and enhanced EMT competency and metastasis, through interaction with specific phosphorylation signals on target proteins. MOE (Min. of Education, S’pore) Published version

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 version

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing. Accepted version

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFβ signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice.

In adult skin wounds, collagen expression rapidly re-establishes the skin barrier, although the resultant scar is aesthetically and functionally inferior to unwounded tissue. Although TGFβ signaling and fibroblasts are known to be responsible for scar-associated collagen production, there are currently no prophylactic treatments for scar management. Fibroblasts in crosstalk with wound keratinocytes orchestrate collagen expression, although the precise paracrine pathways involved remain poorly understood. Herein, we showed that the matricellular protein, angiopoietin-like 4 (ANGPTL4), accelerated wound closure and reduced collagen expression in diabetic and ANGPTL4-knockout mice. Similar observations were made in wild-type rat wounds. Using human fibroblasts as a preclinical model for mechanistic studies, we systematically elucidated that ANGPTL4 binds to cadherin-11, releasing membrane-bound β-catenin which translocate to the nucleus and transcriptionally upregulate the expression of Inhibitor of DNA-binding/differentiation protein 3 (ID3). ID3 interacts with scleraxis, a basic helix-loop-helix transcription factor, to inhibit scar-associated collagen types 1α2 and 3α1 production by fibroblasts. We also showed ANGPTL4 interaction with cadherin-11 in human scar tissue. Our findings highlight a central role for matricellular proteins such as ANGPTL4 in the attenuation of collagen expression and may have a broader implication for other fibrotic pathologies. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

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.

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.

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.

International audience; Although non-melanoma skin cancer (NMSC) is the most common human cancer and its incidence continues to rise worldwide, the mechanisms underlying its development remain incompletely understood. Here, we unveil a cascade of events involving peroxisome proliferator-activated receptor (PPAR) beta/delta and the oncogeneSrc, which promotes the development of ultraviolet (UV)-induced skin cancer in mice. UV-induced PPAR beta/delta activity, which directly stimulatedSrcexpression, increased Src kinase activity and enhanced the EGFR/Erk1/2 signalling pathway, resulting in increased epithelial-to-mesenchymal transition (EMT) marker expression. Consistent with these observations, PPAR beta/delta-null mice developed fewer and smaller skin tumours, and a PPAR beta/delta antagonist prevented UV-dependentSrc stimulation. Furthermore, the expression ofPPAR beta/delta positively correlated with the expression ofSRCand EMT markers in human skin squamous cell carcinoma (SCC), and critically, linear models applied to several human epithelial cancers revealed an interaction between PPAR beta/delta and SRC and TGF beta 1 transcriptional levels. Taken together, these observations motivate the future evaluation of PPAR beta/delta modulators to attenuate the development of several epithelial cancers.

Tumor development and progression to malignancy is increasingly viewed as a consequence of disrupted homeostatic interactions between epithelial cells and the underlying stromal elements. In squamous cell carcinoma of the skin, genomic and histological abnormalities have been observed in keratinocytes and stroma bordering multifocal neoplastic lesions, suggesting a regional carcinogenic signal that promotes aberrant cellular behavior within the tumor microenvironment. This has been termed field cancerization. Cancer associated fibroblasts (CAFs) are the major cell type in the tumor stroma and are known producers of extracellular pro-tumor signaling molecules. While growth factors and cytokines are candidate molecules for targeted therapy, reactive oxygen species (ROS) are gaining attention for their roles in heterotypic paracrine signaling in cancer. The importance of ROS in field cancerization remains poorly understood. Histological analysis of on sections of patient tumor biopsies revealed that ROS was elevated in the tumor stroma. CAFs explanted from patient tumors also released high levels of hydrogen peroxide into the extracellular space. Interestingly, normal fibroblasts exposed to CAF-conditioned medium displayed increased ROS production, an observation that could be reversed by the antioxidant N-acetyl cysteine. Exposure to hydrogen peroxide also resulted in elevated ROS production in normal fibroblasts. Taken together, these findings suggest that CAFs are a potent source of ROS in the tumor microenvironment and can induce a field effect that transforms adjacent normal fibroblasts into a CAF-like state. Transformed fibroblasts in turn produce ROS to spread oxidative stress in the microenvironment, potentially exacerbating field cancerization. Therefore, antioxidant-based therapies targeting the tumor microenvironment may be important adjuvant treatments to current anticancer chemotherapy or targeted therapy.

Background: The role of PPARβ/δ in colorectal cancer has been a hot topic for debate, with both tumor-promoting and anti-tumor roles been reported. Current studies on colorectal cancer have focused on either the whole intestine or on the major tissue type, the intestinal epithelium. In recent years, various studies have emphasized the pro-inflammatory and tumor promoting effects of tumor stroma in various cancers. However, the involvement of tumor stroma in adenocarcinoma development remains a largely unexplored realm of colorectal research. Hence, we hypothesized that PPARB/D expression in colorectal tumor stroma may play an important role in colorectal carcinogenesis. Methods: In this study, we aimed to investigate the role of PPARβ/δ in the colorectal tumor stroma. To study the role of the tumor stroma in vivo, we have adopted a model organism approach involving adult APCmin mice and AOM/DSS-treated wild-type mice models. We collected intestinal adenocarcinomas from these mice and extracted tumor stroma using microscopy-directed laser capture micro-dissection. The collected stroma were recovered for mRNA and analyzed for gene expression levels using real-time PCR. Results and conclusion: We observed contrasting trends in the PPARβ/δ mRNA expression, with elevated levels in stroma of APCmin adenocarcinoma but decreased in AOM/DSS-induced tumors. Our results to date demonstrate the heterogeneity of colorectal tumor stroma and provide an insight to the distinct differences between the two often co-studied murine models of colorectal carcinogenesis. We therefore plan to study if this trend is also observed in human colorectal tumor samples and in murine colorectal cancer models carrying stromal-specific deletion of PPARβ/δ expression. Citation Format: Eddie Han Pin Tan, Ming Keat Sng, Jeremy Soon Kiat Chan, Nguan Soon Tan. Heterogeneity of PPARβ/δ expression in colorectal tumor stroma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2015-3211

The reprogrammed cancer metabolism is an emerging cancer hallmark pervasive in many cancers and is fundamental to neoplastic progression. Many key cancer-promoting pathways are known to converge on the regulation of cellular metabolism. Besides providing basic sustenance to fuel cancer cells limitless replicative potential, emerging data reveals critical roles for the reprogammed metabolism during maglignant progression. Metastasis is often regarded as the end-stage of malignancy and majority of cancer mortality is attributed to the metastatic dissemination of the malignant cancer cells. The initiation of metastasis has been pinpointed as pivotal event, without which, the malignant cancer cells loses their motility and invasive capabilities for distal dissemination. This initiation event highly resembles epithelial-mesenchymal transition (EMT) process that occurs during embryogenesis and wound healing. Microenvironmental signals such as hypoxia and the inflammatory milleu that initiate EMT, interestingly, are also shown to be critical in rewiring cancer metabolism. Thus, it is conceivable that the manipulation of cancer metabolism can impact EMT. Furthermore, whether such metabolic reprogramming is a functional pre-requisite for the initiation of EMT remains unclear. Using three distinct in vitro EMT models, we show that EMT is an energy-demanding process. Comparative microarray analysis of the EMT models reveals that genes involved in fatty acid, glucose and ROS metabolism are altered. We went further to show that cancer cells undergoing EMT, derives much of the ATP/energy from glucose and lipid metabolism. Importantly, we identified several key molecular drivers that coordinate EMT and cellular bioenergetics, which is validated by our in vivo EMT model. Citation Format: Zi Qiang Teo, Ming Keat Sng, Jeremy CHAN, Nguan Soon Tan. Molecular drivers of metabolic reprogramming during EMT. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-198. doi:10.1158/1538-7445.AM2015-LB-198