Publications by authors named "Daniel W Lambert"

43 Publications

Epigenetic modulation of the tumor microenvironment in head and neck cancer: Challenges and opportunities.

Crit Rev Oncol Hematol 2021 Jun 17;164:103397. Epub 2021 Jun 17.

The Brazilian Bioscience National Laboratory, Center for Research in Energy and Materials, Campinas, Brazil.

Head and neck cancer is globally challenging due to the resistance to therapy and aggressive behavior leading to high rates of mortality. Recent findings show that the tumor microenvironment plays a role in the maintenance and progression of many solid tumors, including head and neck cancer. The mechanisms involved in the modulation and regulation of the tumor microenvironment remain poorly understood. Increasing evidence suggests that epigenetic events can modulate the crosstalk between neoplastic and non-neoplastic cells during tumor progression. In this review, we explore the current understanding of the involvement of epigenetic events in the modulation of the tumor microenvironment and its impact on head and neck cancer behavior. We also explore the latest therapeutic strategies that use epigenetic-modulating drugs to manage tumor growth and progression.
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http://dx.doi.org/10.1016/j.critrevonc.2021.103397DOI Listing
June 2021

Understanding fibroblast behaviour in 3D biomaterials.

Tissue Eng Part B Rev 2021 Jun 9. Epub 2021 Jun 9.

University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom of Great Britain and Northern Ireland;

Traditional monolayer culture fails to fully recapitulate the in vivo environment of connective tissue cells such as the fibroblast. When cultured on stiff two-dimensional plastic, fibroblasts become highly proliferative forming broad lamellipodia and stress fibres. Conversely, in different 3D culture systems fibroblasts have displayed a diverse array of features; from an 'activated' phenotype like that observed in 2D cultures and by myofibroblasts, to a quiescent state that likely better represents in vivo fibroblasts at rest. Today, a plethora of microfabrication techniques have made 3D culture commonplace, for both tissue engineering purposes and in the study of basic biological interactions. However, establishing the in vivo mimetic credentials of different biomimetic materials is not always straightforward, particularly in the context of fibroblast responses. Fibroblast behaviour is governed by the complex interplay of biological features such as integrin binding sites, material mechanical properties which influence cellular mechanotransduction and microarchitectural features like pore and fibre size as well as chemical cues. Furthermore, fibroblasts are a heterogeneous group of cells with specific phenotypic traits dependent on their tissue of origin. These features have made understanding the influence of biomaterials on fibroblast behaviour a challenging task. Here we present a review of the strategies used to investigate fibroblast behaviour with a focus on the material properties that influence fibroblast activation, a process which becomes pathological in fibrotic diseases and certain cancers.
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http://dx.doi.org/10.1089/ten.TEB.2021.0010DOI Listing
June 2021

The role of icIL-1RA in keratinocyte senescence and development of the senescence-associated secretory phenotype.

J Cell Sci 2021 02 22;134(4). Epub 2021 Feb 22.

Unit of Oral and Maxillofacial Medicine, Pathology and Surgery, University of Sheffield, Sheffield S10 2TA, UK

There is compelling evidence that senescent cells, through the senescence-associated secretory phenotype (SASP), can promote malignant transformation and invasion. Interleukin-1 (IL-1) is a key mediator of this cytokine network, but the control of its activity in the senescence programme has not been elucidated. IL-1 signalling is regulated by IL-1RA, which has four variants. Here, we show that expression of intracellular IL-1RA type 1 (icIL-1RA1), which competitively inhibits binding of IL-1 to its receptor, is progressively lost during oral carcinogenesis and that the pattern of expression is associated with keratinocyte replicative fate We demonstrate that icIL-1RA1 is an important regulator of the SASP in mortal cells, as CRISPR/Cas9-mediated icIL-1RA1 knockdown in normal and mortal dysplastic oral keratinocytes is followed by increased IL-6 and IL-8 secretion, and rapid senescence following release from RhoA-activated kinase inhibition. Thus, we suggest that downregulation of icIL-1RA1 in early stages of the carcinogenesis process can enable the development of a premature and deregulated SASP, creating a pro-inflammatory state in which cancer is more likely to arise.
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http://dx.doi.org/10.1242/jcs.252080DOI Listing
February 2021

The Emerging Potential of Extracellular Vesicles in Cell-Free Tissue Engineering and Regenerative Medicine.

Tissue Eng Part B Rev 2020 Dec 3. Epub 2020 Dec 3.

School of Clinical Dentistry, The University of Sheffield, Sheffield, United Kingdom.

Extracellular vesicles (Evs) are membrane-enclosed vesicles secreted by all cell types that mediate cell-cell communication via their protein, lipid, carbohydrate, and nucleic acid (RNA, DNA) cargo. EVs are involved in a multitude of physiological processes, including development, cell differentiation, and angiogenesis, and have been implicated in tissue repair. Thus, they have been suggested to offer opportunities for the development of novel cell-free tissue engineering (TE) approaches. In this review, we provide an overview of current understanding and emerging applications of EVs in TE and address opportunities and challenges for clinical translation. In addition, we discuss systemic and local routes of delivery of EVs and the advantages and disadvantages of different biomaterials in providing a substrate for the sustained release of EVs . Impact statement Extracellular vesicles (EVs) are nanoscale, membrane-bound vesicles released by most, if not all, cells in the body. They are implicated in a wide range of physiological processes and diseases ranging from cancer to neurodegeneration, and hold huge potential as mediators of tissue regeneration. This has led to an explosion of interest in using EVs in a variety of tissue engineering applications. In this review, we provide an overview of current progress in the field and highlight the opportunities and challenges of harnessing the potential of EVs in regenerative medicine.
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http://dx.doi.org/10.1089/ten.TEB.2020.0222DOI Listing
December 2020

Extracellular vesicles and the extracellular matrix: a new paradigm or old news?

Biochem Soc Trans 2020 10;48(5):2335-2345

School of Clinical Dentistry, University of Sheffield, Sheffield, U.K.

Extracellular vesicles (EV) are implicated in a variety of functions affecting the extracellular matrix (ECM), including matrix degradation, cross-linking of matrix proteins and matrix calcification. These processes are important in many physiological contexts such as angiogenesis and wound healing, and dysregulation of ECM homeostasis contributes to a wide range of diseases including fibrosis, cancer and arthritis. Most studies of EV have focussed on their roles in cell:cell communication, but EV can exist as integral components of the ECM. By far the most well-characterised ECM-resident EV are matrix vesicles (MV) in bone, but the broader role of EV in the ECM is not well understood. This review will explore what is known of the roles of EV in the ECM and will also highlight the similarities and differences between MV and other EV.
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http://dx.doi.org/10.1042/BST20200717DOI Listing
October 2020

ROCK inhibition modulates the senescence-associated secretory phenotype (SASP) in oral keratinocytes.

FEBS Open Bio 2020 12 6;10(12):2740-2749. Epub 2020 Nov 6.

Unit of Oral and Maxillofacial Medicine, Pathology and Surgery, University of Sheffield, Sheffield, UK.

Senescent cells accumulate in different organs and develop a senescence-associated secretory phenotype (SASP), associated with the development of age-related pathologies. The constitution of the SASP varies among cell types and with the method of senescence induction; nevertheless, there is substantial overlap among SASPs, especially the presence of pro-inflammatory cytokines such as IL-1β, IL-1α, IL-6 and IL-8. These cytokines are highly conserved among SASPs and are implicated in the development of several cancers. Here, we report that ROCK inhibition by Y-27632 reduces levels of IL-1α, IL-1β, IL-6 and IL-8 secreted by senescent normal and dysplastic oral keratinocytes without affecting the permanent cell growth arrest. The data indicate some inflammatory genes downregulated by Y-27632 remain downregulated even after repeated passage in the absence of Y-27632. We propose ROCK kinase inhibition as a novel alternative to current strategies to modulate the inflammatory components of the SASP, without compromising the permanent cell growth arrest. This observation potentially has wide clinical applications, given the involvement of senescence in cancer and a wide range of age-related disease. It also suggests care should be exercised when using Y-27632 to facilitate cell expansion of primary cells, as its effects on gene expression are not entirely reversible.
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http://dx.doi.org/10.1002/2211-5463.13012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714064PMC
December 2020

Discovery and characterization of ACE2 - a 20-year journey of surprises from vasopeptidase to COVID-19.

Clin Sci (Lond) 2020 09;134(18):2489-2501

School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT,U.K.

Angiotensin-converting enzyme (ACE) is a zinc membrane metallopeptidase that plays a key role in regulating vasoactive peptide levels and hence cardiovascular activity through its conversion of angiotensin I (Ang I) to Ang II and its metabolism of bradykinin. The discovery of its homologue, ACE2, 20 years ago has led to intensive comparisons of these two enzymes revealing surprising structural, catalytic and functional distinctions between them. ACE2 plays multiple roles not only as a vasopeptidase but also as a regulator of amino acid transport and serendipitously as a viral receptor, mediating the cellular entry of the coronaviruses causing severe acute respiratory syndrome (SARS) and, very recently, COVID-19. Catalytically, ACE2 functions as a monocarboxypeptidase principally converting the vasoconstrictor angiotensin II to the vasodilatory peptide Ang-(1-7) thereby counterbalancing the action of ACE on the renin-angiotensin system (RAS) and providing a cardioprotective role. Unlike ACE, ACE2 does not metabolise bradykinin nor is it inhibited by classical ACE inhibitors. However, it does convert a number of other regulatory peptides in vitro and in vivo. Interest in ACE2 biology and its potential as a possible therapeutic target has surged in recent months as the COVID-19 pandemic rages worldwide. This review highlights the surprising discoveries of ACE2 biology during the last 20 years, its distinctions from classical ACE and the therapeutic opportunities arising from its multiple biological roles.
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http://dx.doi.org/10.1042/CS20200476DOI Listing
September 2020

Oral cancer in Papua New Guinea: looking back and looking forward.

Oral Surg Oral Med Oral Pathol Oral Radiol 2020 Sep 20;130(3):292-297. Epub 2020 Jun 20.

School of Clinical Dentistry, University of Sheffield, Sheffield, UK. Electronic address:

Oral diseases, including cancers, affect 3.5 billion people globally and remain largely untreated in low- to middle-income countries because of lack of resources. In Papua New Guinea (PNG), oral cancer has, for many decades, been identified as the most common cancer in men, but as the GLOBOCAN 2018 data are estimates extrapolated from surrounding countries, the real prevalence of this disease is not known. The PNG National Health Plan (2011-2020) highlights the need to improve health care, but oral health is not identified as a priority. Alcohol, tobacco, and areca nut/betel quid, which are the social and commercial determinants of oral cancer, are common risk factors, and there are robust data linking these risk factors to oral cancer in PNG. Our recent Global Challenges Research Fund Workshop on Oral Cancer, held in Port Moresby, PNG, brought together a number of researchers in oral cancer epidemiology and translational science with clinicians from PNG to assess the current situation and plan ways to move forward. In this article, we will review the literature on oral cancer in PNG, and make suggestions as to how, collaboratively, we can address the issues identified, ultimately, for the benefit of the people of PNG.
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http://dx.doi.org/10.1016/j.oooo.2020.06.010DOI Listing
September 2020

Oral cancer stem cells drive tumourigenesis through activation of stromal fibroblasts.

Oral Dis 2020 Jun 27. Epub 2020 Jun 27.

School of Clinical Dentistry, University of Sheffield, Sheffield, UK.

Background: Cancer stem cells are responsible for tumour progression and chemoresistance. Fibroblasts surrounding a tumour also promote progression and fibroblast "activation" is an independent prognostic marker in oral cancer. Cancer stem cells may therefore promote tumourigenesis through communication with stromal fibroblasts.

Methods: Cancer stem cells were isolated from oral cancer cell lines by adherence to fibronectin or cisplatin resistance. Fibroblasts were exposed to conditioned medium from these cells, and the activation markers, alpha smooth muscle actin and interleukin-6, were assessed using qPCR and immunofluorescence. Stem cell markers and smooth muscle actin were examined in oral cancer tissue using immunohistochemistry.

Results: Adherent and chemoresistant cells expressed increased levels of stem cell markers CD24, CD44 and CD29 compared with unsorted cells. Adherent cells exhibited lower growth rate, higher colony forming efficiency and increased cisplatin resistance than unsorted cells. Smooth muscle actin and Interleukin-6 expression were increased in fibroblasts exposed to conditioned medium. In oral cancer tissue, there was a positive correlation between expression of αSMA and stem cell markers.

Conclusions: Adherence to fibronectin and chemoresistance isolates stem-like cells that can activate fibroblasts, which together with a correlation between markers of both in vivo, provides a mechanism by which such cells drive tumourigenesis.
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http://dx.doi.org/10.1111/odi.13513DOI Listing
June 2020

Comprehensive functional profiling of long non-coding RNAs through a novel pan-cancer integration approach and modular analysis of their protein-coding gene association networks.

BMC Genomics 2019 Jun 3;20(1):454. Epub 2019 Jun 3.

Sheffield Institute for Nucleic Acids (SInFoNiA), Department of Oncology and Metabolism, University of Sheffield, Sheffield, South Yorkshire, UK.

Background: Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cellular processes in diseases such as cancer, although the functions of most remain poorly understood. To address this, here we apply a novel strategy to integrate gene expression profiles across 32 cancer types, and cluster human lncRNAs based on their pan-cancer protein-coding gene associations. By doing so, we derive 16 lncRNA modules whose unique properties allow simultaneous inference of function, disease specificity and regulation for over 800 lncRNAs.

Results: Remarkably, modules could be grouped into just four functional themes: transcription regulation, immunological, extracellular, and neurological, with module generation frequently driven by lncRNA tissue specificity. Notably, three modules associated with the extracellular matrix represented potential networks of lncRNAs regulating key events in tumour progression. These included a tumour-specific signature of 33 lncRNAs that may play a role in inducing epithelial-mesenchymal transition through modulation of TGFβ signalling, and two stromal-specific modules comprising 26 lncRNAs linked to a tumour suppressive microenvironment and 12 lncRNAs related to cancer-associated fibroblasts. One member of the 12-lncRNA signature was experimentally supported by siRNA knockdown, which resulted in attenuated differentiation of quiescent fibroblasts to a cancer-associated phenotype.

Conclusions: Overall, the study provides a unique pan-cancer perspective on the lncRNA functional landscape, acting as a global source of novel hypotheses on lncRNA contribution to tumour progression.
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http://dx.doi.org/10.1186/s12864-019-5850-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547491PMC
June 2019

Extranodal extension in oral cancer: A role for the nodal microenvironment?

J Oral Pathol Med 2019 Nov 21;48(10):863-870. Epub 2019 May 21.

Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, UK.

Oral squamous cell carcinoma (OSCC) is a significant cause of morbidity and mortality worldwide and accounts for the majority of head and neck cancers. Metastasis of primary tumours, primarily to cervical lymph nodes in the neck, is associated with worsening prognosis. Furthermore, the prognosis of patients with extranodal extension of metastatic tumour from the lymph nodes into the neck tissues is particularly poor. The factors affecting this process are poorly understood, and detection is difficult pre-surgery. Mounting evidence shows that components of the tumour microenvironment including cancer-associated fibroblasts, vascular and lymphatic endothelial cells, the extracellular matrix and inflammatory immune cells, are important modulators of tumour behaviour in primary OSCC and other cancers. However, little is known about the lymph node microenvironment, its response to tumour presence and role in extranodal extension. In addition, there are many lymph node-specific cell types and structures, such as fibroblast reticular cells and high endothelial venules, making the lymph node microenvironment distinct from that found at primary tumour sites, and which contribute to the nodal response to tumour presence. This review details the current knowledge regarding the lymph node tumour microenvironment in OSCC and its role in lymph node metastasis and extranodal extension and relates this to features of the primary tumour. Understanding the role that the lymph node microenvironment plays in promoting tumour development and extranodal extension may aid the identification of novel biomarkers and alternative treatment strategies to improve the prognosis of patients with advanced OSCC.
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http://dx.doi.org/10.1111/jop.12870DOI Listing
November 2019

Extracellular vesicles: translational challenges and opportunities.

Biochem Soc Trans 2018 10 20;46(5):1073-1082. Epub 2018 Sep 20.

Academic Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, S10 2TA Sheffield, U.K.

Extracellular vesicles (EVs) are a heterogeneous group of small lipid-enclosed structures with myriad roles in physiology and disease. The recent surge of interest in EVs has led to greater understanding of their biology and appreciation of how they might be utilised as diagnostic and therapeutic tools. There remain, however, a number of challenges that must be overcome before EVs may be used routinely in the clinic. In this review we will discuss the translational potential of EVs and the current technologies available to isolate, purify and analyse EVs and their contents.
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http://dx.doi.org/10.1042/BST20180112DOI Listing
October 2018

Targeting HOX-PBX interactions causes death in oral potentially malignant and squamous carcinoma cells but not normal oral keratinocytes.

BMC Cancer 2018 Jul 6;18(1):723. Epub 2018 Jul 6.

Integrated Biosciences, School of Clinical Dentistry, University of Sheffield, Sheffield, UK.

Background: High HOX gene expression has been described in many cancers, including oral squamous cell carcinoma and the functional roles of these genes are gradually being understood. The pattern of overexpression suggests that inhibition may be useful therapeutically. Inhibition of HOX protein binding to PBX cofactors by the use of synthetic peptides, such as HXR9, results in apoptosis in multiple cancers.

Methods: Activity of the HOX-PBX inhibiting peptide HXR9 was tested in immortalised normal oral (NOK), potentially-malignant (PMOL) and squamous cell carcinoma (OSCC) cells, compared to the inactive peptide CXR9. Cytotoxicity was assessed by LDH assay. Expression of PBX1/2 and c-Fos was assessed by qPCR and western blotting. Apoptosis was assessed by Annexin-V assay.

Results: PMOL and OSCC cells expressed PBX1/2. HOX-PBX inhibition by HXR9 caused death of PMOL and OSCC cells, but not NOKs. HXR9 treatment resulted in apoptosis and increased expression of c-Fos in some cells, whereas CXR9 did not. A correlation was observed between HOX expression and resistance to HXR9.

Conclusion: Inhibition of HOX-PBX interactions causes selective apoptosis of OSCC/PMOL, indicating selective toxicity that may be useful clinically.
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http://dx.doi.org/10.1186/s12885-018-4622-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035449PMC
July 2018

A miRNA-145/TGF-β1 negative feedback loop regulates the cancer-associated fibroblast phenotype.

Carcinogenesis 2018 05;39(6):798-807

Integrated Biosciences, School of Clinical Dentistry, Sheffield, UK.

The dissemination of cancer cells to local and distant sites depends on a complex and poorly understood interplay between malignant cells and the cellular and non-cellular components surrounding them, collectively termed the tumour microenvironment. One of the most abundant cell types of the tumour microenvironment is the fibroblast, which becomes corrupted by locally derived cues such as TGF-β1 and acquires an altered, heterogeneous phenotype (cancer-associated fibroblasts, CAF) supportive of tumour cell invasion and metastasis. Efforts to develop new treatments targeting the tumour mesenchyme are hampered by a poor understanding of the mechanisms underlying the development of CAF. Here, we examine the contribution of microRNA to the development of experimentally-derived CAF and correlate this with changes observed in CAF derived from tumours. Exposure of primary normal human fibroblasts to TGF-β1 resulted in the acquisition of a myofibroblastic CAF-like phenotype. This was associated with increased expression of miR-145, a miRNA predicted in silico to target multiple components of the TGF-β signalling pathway. miR-145 was also overexpressed in CAF derived from oral cancers. Overexpression of miR-145 blocked TGF-β1-induced myofibroblastic differentiation and reverted CAF towards a normal fibroblast phenotype. We conclude that miR-145 is a key regulator of the CAF phenotype, acting in a negative feedback loop to dampen acquisition of myofibroblastic traits, a key feature of CAF associated with poor disease outcome.
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http://dx.doi.org/10.1093/carcin/bgy032DOI Listing
May 2018

Extracellular vesicles in the tumour microenvironment.

Philos Trans R Soc Lond B Biol Sci 2018 01;373(1737)

School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, UK

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http://dx.doi.org/10.1098/rstb.2016.0475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717432PMC
January 2018

HPV-negative, but not HPV-positive, oropharyngeal carcinomas induce fibroblasts to support tumour invasion through micro-environmental release of HGF and IL-6.

Carcinogenesis 2018 02;39(2):170-179

School of Clinical Dentistry, University of Sheffield, Sheffield, South Yorkshire, UK.

Human papillomavirus (HPV) infection is causally related to a subset of oropharyngeal carcinomas (OPC) and is linked to a more favourable prognosis compared to HPV-negative OPC. The mechanisms underlying this effect on prognosis are not fully understood, but interactions with the tumour microenvironment may be pivotal. Here, we investigated the role of the tumour microenvironment in HPV-positive compared to HPV-negative cancer using 2D and 3D modelling of OPC interactions with stromal fibroblasts. HPV-negative, but not HPV-positive, OPC-derived cell lines induced a rapid fibroblast secretory response that supported 2D cancer cell migration and invasion in vitro. Array profiling of this HPV-negative induced fibroblast secretome identified hepatocyte growth factor (HGF) as the principal secreted factor that promoted cancer cell migration. The interaction between HPV-negative cell lines and fibroblasts in 2D was prevented using c-Met (HGF receptor) inhibitors, which further restricted both HPV-negative and positive cell invasion in 3D co-culture models. Furthermore, we discovered a synergistic relationship between HGF and IL-6 in the support of migration that relates JAK activation to HGF responsiveness in HPV-negative lines. In summary, our data show significant differences in the interactions between HPV-positive and HPV-negative OPC cells and stromal fibroblasts. In addition, we, provide in vitro evidence to support the clinical application of c-MET inhibitors in the control of early HPV-negative OPC.
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http://dx.doi.org/10.1093/carcin/bgx130DOI Listing
February 2018

Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma.

Oncotarget 2017 Sep 19;8(43):74736-74754. Epub 2017 Aug 19.

Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba, SP, Brazil.

Oral squamous cell carcinoma (OSCC) prognosis is related to clinical stage and histological grade. However, this stratification needs to be refined. We conducted a comparative proteome study in microdissected samples from normal oral mucosa and OSCC to identify biomarkers for malignancy. Fascin and plectin were identified as differently expressed and both are implicated in several malignancies, but the clinical impacts of aberrant fascin and plectin expression in OSCCs remains largely unknown. Immunohistochemistry and real-time quantitative PCR were carried out in OSCC samples and cell lines. A loss-of-function strategy using shRNA targeting fascin was employed to investigate and the fascin role on oral tumorigenesis. Transfections of microRNA mimics were performed to determine whether the fascin overexpression is regulated by miR-138 and miR-145. We found that fascin and plectin are frequently upregulated in OSCC samples and cell lines, but only fascin overexpression is an independent unfavorable prognostic indicator of disease-specific survival. In combination with advanced T stage, high fascin level is also an independent factor of disease-free survival. Knockdown of fascin in OSCC cells promoted cell adhesion and inhibited migration, invasion and EMT, and forced expression of miR-138 in OSCC cells significantly decreased the expression of fascin. In addition, fascin downregulation leads to reduced filopodia formation and decrease on paxillin expression. The subcutaneous xenograft model showed that tumors formed in the presence of low levels of fascin were significantly smaller compared to those formed with high fascin levels. Collectively, our findings suggest that fascin expression correlates with disease progression and may serve as a prognostic marker and therapeutic target for patients with OSCC.
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http://dx.doi.org/10.18632/oncotarget.20360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650375PMC
September 2017

Prognostic value of the immunohistochemical detection of cancer-associated fibroblasts in oral cancer: A systematic review and meta-analysis.

J Oral Pathol Med 2018 May 5;47(5):443-453. Epub 2017 Sep 5.

Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba-SP, Brazil.

Aim: To perform a meta-analysis to assess whether the presence of cancer-associated fibroblasts (CAF) is a prognostic marker of oral squamous cell carcinomas (OSCC).

Methods: Immunohistochemical studies assessing the prognostic relevance of CAF (alpha smooth muscle actin (α-SMA)-positive fibroblasts) in patients with OSCC were systematically reviewed using Cochrane, Lilacs, PubMed, Scopus, and Web of Science databases. The outcomes assessed were overall survival (OS) and disease-free survival (DFS). The meta-analysis was performed using the random- and fixed-effects model with adjusted hazard ratio (HR) and 95% confidence intervals (95% CI) as effect measures. The methodological quality of the included studies was assessed using the Meta-Analysis of Statistics Assessment and Review Instrument (MAStARI) tool, and the evidence quality was assessed by the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) system.

Results: The presence of high levels of CAF in the stroma of OSCC predicted shortened time to DFS (HR = 3.32, 95% CI: 2.09-5.26, P < .00001) and an overall decrease in survival (HR: 2.16, 95% CI: 1.60-2.92, P < .00001). Moreover, high presence of CAF was frequently reported in association with parameters that worsen the prognosis in OSCC, including advanced disease stage (TNM classification), recurrence, tumor grade, depth of invasion, vascular, lymphatic and neural invasion, and extranodal metastatic spread.

Conclusion: The presence of CAF, as assessed by α-SMA-positive fibroblasts in the stroma, indicates poor prognosis in patients with OSCC.
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http://dx.doi.org/10.1111/jop.12623DOI Listing
May 2018

Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that differentially impacts on cancer prognosis.

Aging (Albany NY) 2016 12;9(1):114-132

Cancer Sciences Unit, Faculty of Medicine, University of Southampton, SO166YD, UK.

Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured , showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. , we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-β signaling. Similar to TGF-β1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes.
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http://dx.doi.org/10.18632/aging.101127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310659PMC
December 2016

HOPX functions as a tumour suppressor in head and neck cancer.

Sci Rep 2016 12 9;6:38758. Epub 2016 Dec 9.

Department of Oral and Craniofacial Sciences and Oral Cancer Research and Coordinating Centre, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Head and neck squamous cell carcinoma (HNSCC) is generalized term that encompasses a diverse group of cancers that includes tumours of the oral cavity (OSCC), oropharynx (OPSCC) and nasopharynx (NPC). Genetic alterations that are common to all HNSCC types are likely to be important for squamous carcinogenesis. In this study, we have investigated the role of the homeodomain-only homeobox gene, HOPX, in the pathogenesis of HNSCC. We show that HOPX mRNA levels are reduced in OSCC and NPC cell lines and tissues and there is a general reduction of HOPX protein expression in these tumours and OPSCCs. HOPX promoter methylation was observed in a subset of HNSCCs and was associated with a worse overall survival in HPV negative tumours. RNAseq analysis of OSCC cells transfected with HOPX revealed a widespread deregulation of the transcription of genes related to epithelial homeostasis and ectopic over-expression of HOPX in OSCC and NPC cells inhibited cell proliferation, plating efficiency and migration, and enhanced sensitivity to UVA-induced apoptosis. Our results demonstrate that HOPX functions as a tumour suppressor in HNSCC and suggest a central role for HOPX in suppressing epithelial carcinogenesis.
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http://dx.doi.org/10.1038/srep38758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146930PMC
December 2016

Cancer-associated fibroblasts - Not-so-innocent bystanders in metastasis to bone?

J Bone Oncol 2016 Sep 16;5(3):128-131. Epub 2016 May 16.

Integrated Biosciences, School of Clinical Dentistry, University of Sheffield, S10 2TA, United Kingdom.

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http://dx.doi.org/10.1016/j.jbo.2016.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063218PMC
September 2016

Physiological Fluid Flow Moderates Fibroblast Responses to TGF-β1.

J Cell Biochem 2017 04 10;118(4):878-890. Epub 2016 Nov 10.

Academic Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, UK.

Fibroblasts are the major cellular component of connective tissue and experience mechanical perturbations due to matrix remodelling and interstitial fluid movement. Transforming growth factor β1 (TGF-β1) can promote differentiation of fibroblasts in vitro to a contractile myofibroblastic phenotype characterised by the presence of α-smooth muscle actin (α-SMA) rich stress fibres. To study the role of mechanical stimulation in this process, we examined the response of primary human fibroblasts to physiological levels of fluid movement and its influence on fibroblast differentiation and responses to TGF-β1. We reported that in both oral and dermal fibroblasts, physiological levels of fluid flow induced widespread changes in gene expression compared to static cultures, including up-regulation of genes associated with TGFβ signalling and endocytosis. TGF-β1, activin A and markers of myofibroblast differentiation including α-SMA and collagen IA1 were also increased by flow but surprisingly the combination of flow and exogenous TGF-β1 resulted in reduced differentiation. Our findings suggest this may result from enhanced internalisation of caveolin and TGF-β receptor II. These findings suggest that a) low levels of fluid flow induce myofibroblast differentiation and b) fluid flow antagonises the fibroblast response to pro-differentiation signals such as TGF-β1. We propose that this may be a novel mechanism by which mechanical forces buffer responses to chemical signals in vivo, maintaining a context-specific fibroblast phenotype. J. Cell. Biochem. 118: 878-890, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcb.25767DOI Listing
April 2017

A miR-335/COX-2/PTEN axis regulates the secretory phenotype of senescent cancer-associated fibroblasts.

Aging (Albany NY) 2016 08;8(8):1608-35

Integrated Biosciences, School of Clinical Dentistry, University of Sheffield, S10 2TA, UK.

Senescent cancer-associated fibroblasts (CAF) develop a senescence-associated secretory phenotype (SASP) that is believed to contribute to cancer progression. The mechanisms underlying SASP development are, however, poorly understood. Here we examined the functional role of microRNA in the development of the SASP in normal fibroblasts and CAF. We identified a microRNA, miR-335, up-regulated in the senescent normal fibroblasts and CAF and able to modulate the secretion of SASP factors and induce cancer cell motility in co-cultures, at least in part by suppressing the expression of phosphatase and tensin homologue (PTEN). Additionally, elevated levels of cyclo-oxygenase 2 (PTGS2; COX-2) and prostaglandin E2 (PGE2) secretion were observed in senescent fibroblasts, and inhibition of COX-2 by celecoxib reduced the expression of miR-335, restored PTEN expression and decreased the pro-tumourigenic effects of the SASP. Collectively these data demonstrate the existence of a novel miRNA/PTEN-regulated pathway modulating the inflammasome in senescent fibroblasts.
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http://dx.doi.org/10.18632/aging.100987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032686PMC
August 2016

The role of HOX genes in head and neck squamous cell carcinoma.

J Oral Pathol Med 2016 Apr 14;45(4):239-47. Epub 2015 Dec 14.

Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, UK.

Recent decades have witnessed the publication of numerous studies reporting alterations in the genome and transcriptome of head and neck squamous cell carcinoma (HNSCC). Currently, the utilisation of these alterations as biomarkers and targets for therapy is limited and new, useful molecular characteristics are being sought. Many of the published HNSCC gene expression profiles demonstrate alterations in the expression of HOX genes. These are a family of Homeobox-containing genes which are involved in developmental patterning and morphogenesis in the embryo, and which are often aberrantly expressed in cancer. The 39 HOX genes found in the human genome are arranged in four paralogous groups at different chromosomal loci. These control a wide range of cellular processes, including proliferation and migration, which are relevant in the context of cancer development. In this review article, we will outline the biology of HOX genes in relation to cancer and summarise the accumulating evidence for their role in the development of HNSCC and the possibility that they could be a therapeutic target in this malignancy. We will also identify areas where our current understanding is weak to focus future work and appraise the ongoing strategies for pharmacological intervention.
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http://dx.doi.org/10.1111/jop.12388DOI Listing
April 2016

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis.

PLoS One 2015 28;10(8):e0136599. Epub 2015 Aug 28.

Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba-SP, Brazil.

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136599PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552554PMC
May 2016

The role of HOXB9 and miR-196a in head and neck squamous cell carcinoma.

PLoS One 2015 10;10(4):e0122285. Epub 2015 Apr 10.

Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, United Kingdom; Department of Oral Pathology and Biology, University of Pretoria, Pretoria, South Africa.

Background: Previous studies have demonstrated that a number of HOX genes, a family of transcription factors with key roles in early development, are up-regulated in head and neck squamous cell carcinoma (HNSCC) and other cancers. The loci of several Homeobox (HOX) genes also contain microRNAs (miRs), including miR-196a.

Methods: Global miR expression and expression of all 39 HOX genes in normal oral keratinocytes (NOKs), oral pre-malignant (OPM) and HNSCC cells was assessed by expression microarray and qPCR and in tissues by immunohistochemistry (IHC) and qPCR of laser microdissected (LCM) tissues. Expression of miR196a and HOXB9 was reduced using anti-miR-196a and siRNA, respectively. Expression microarray profiles of anti-miR196a and pre-miR196a transfected cells were compared to parental cells in order to identify novel targets of miR-196a. Putative miR196a targets were validated by qPCR and were confirmed as binding to the 3'UTR of miR196a by a dual luciferase reporter assay combined with mutational analysis of the miR-196a binding site.

Results: miR-196a and HOXB9 are highly expressed in HNSCC compared to NOKs, a pattern also seen in HNSCC tissues by HOXB9 IHC and qPCR of miR-196a in LCM tissue. Knock-down of miR-196a expression decreased HNSCC cell migration, invasion and adhesion to fibronectin, but had no effect on proliferation. Furthermore, knock-down of HOXB9 expression decreased migration, invasion and proliferation but did not alter adhesion. We identified a novel primary mRNA transcript containing HOXB9 and miR196a-1 as predicted from in-silico analysis. Expression array analysis identified a number of miR196a targets, including MAMDC2 and HOXC8. We confirmed that MAMDC2 is a novel miR-196a target using a dual luciferase reporter assay with the effect abolished on mutation of the binding site.

Conclusions: These results show that miR-196a and HOXB9 are overexpressed, perhaps co-ordinately, as HNSCC develops and exert a pro-tumourigenic phenotype in HNSCC and OPM cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122285PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393232PMC
March 2016

Angiotensin-converting enzyme 2 is subject to post-transcriptional regulation by miR-421.

Clin Sci (Lond) 2014 Aug;127(4):243-9

†School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K.

ACE2 (angiotensin converting enzyme 2) plays a critical role in the local tissue RAS (renin-angiotensin system) by hydrolysing the potent hypertensive and mitogenic peptide AngII (angiotensin II). Changes in the levels of ACE2 have been observed in a number of pathologies, including cardiovascular disease, but little is known of the mechanisms regulating its expression. In the present study, therefore, the potential role of miRNAs in the regulation of ACE2 expression in primary human cardiac myofibroblasts was examined. Putative miRNA-binding sites were identified in the 3'-UTR of the ACE2 transcript using online prediction algorithms. Two of these, miR-200b and miR-421, were selected for further analysis. A reporter system using the 3'-UTR of ACE2 fused to the coding region of firefly luciferase was used to determine the functionality of the identified binding sites in vitro. This identified miR-421, but not miR-200b, as a potential regulator of ACE2. The ability of miR-421, an miRNA implicated in the development of thrombosis, to down-regulate ACE2 expression was subsequently confirmed by Western blot analysis of both primary cardiac myofibroblasts and transformed cells transfected with a synthetic miR-421 precursor. Real-time PCR analysis of miR-421 revealed widespread expression in human tissues. miR-421 levels in cardiac myofibroblasts showed significant inter-patient variability, in keeping with the variability of ACE2 expression we have observed previously. In conclusion, the present study is the first to demonstrate that ACE2 may be subject to post-transcriptional regulation and reveals a novel potential therapeutic target, miR-421, which could be exploited to modulate ACE2 expression in disease.
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http://dx.doi.org/10.1042/CS20130420DOI Listing
August 2014

Endothelin-converting enzyme-1 (ECE-1) is post-transcriptionally regulated by alternative polyadenylation.

PLoS One 2014 31;9(1):e83260. Epub 2014 Jan 31.

Integrated Biosciences, School of Clinical Dentistry, University of Sheffield, Sheffield, United Kingdom ; CR-UK/YCR Sheffield Cancer Centre, University of Sheffield, Sheffield, United Kingdom.

Endothelin-converting enzyme-1 (ECE-1) is the enzyme predominantly responsible for producing active endothelin-1 (ET-1), a mitogenic peptide implicated in the aetiology of a number of diseases, including cancer. Elevated levels of ECE-1 have been observed in a range of malignancies, with high expression conferring poor prognosis and aiding the acquisition of androgen independence in prostate cancer. The mechanisms regulating the expression of ECE-1 in cancer cells are poorly understood, hampering the development of novel therapies targeting the endothelin axis. Here we provide evidence that the expression of ECE-1 is markedly inhibited by its 3'UTR, and that alternative polyadenylation (APA) results in the production of ECE-1 transcripts with truncated 3'UTRs which promote elevated protein expression. Abolition of the ECE-1 APA sites reduced protein expression from a reporter vector in prostate cancer cells, suggesting these sites are functional. This is the first study to identify ECE-1 as a target for APA, a regulatory mechanism aberrantly activated in cancer cells, and provides novel information about the mechanisms leading to ECE-1 overexpression in malignant cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083260PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908869PMC
October 2014