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Supplementary Methods, Table 1, Figures 1-4 from Induction of Human Epithelial Stem/Progenitor Expansion by FOXM1

In advanced metastatic cancers with reduced patient survival and poor prognosis, expression of vimentin, a type III intermediate filament protein is frequently observed. Vimentin appears to suppress epithelial characteristics and augments cell migration but the molecular basis for these changes are not well understood. Here we have ectopically expressed vimentin in MCF-7 and investigated its genomic and functional implications. Vimentin changed the cell shape, by decreasing major axis and major axis angle, and increased cell migration, without affecting proliferation. Vimentin downregulated major keratin genes KRT8, KRT18 and KRT19. Transcriptome-coupled GO and KEGG analyses revealed that vimentin-affected genes were linked to either cell-cell/cell-ECM or cell cycle/proliferation specific pathways. Using shRNA mediated knockdown of vimentin in two breast cancer cell types; MCF-7FV (ectopically expressing) and MDA-MB-231 (endogenously expressing), we identified a vimentin-specific signature consisting of 13 protein encoding genes (CDH5, AXL, PTPRM, TGFBI, CDH10, FOXM1, BCL2, NES, E2F1, FOXM1, CDC45, FSD1, BCL2, KIF26A and WISP2) and two long non-coding RNAs, LINC00052 and C15ORF9-AS1. CDH5, an endothelial cadherin, which mediates cell-cell junctions was the most downregulated protein encoding gene. Interestingly, downregulation of CDH5 by shRNA significantly increased cell migration confirming our RNA-Seq data. Furthermore, vimentin reduced MCF-7 nuclear area perhaps through altered lamin expression. Collectively, we demonstrate, for the first time, that vimentin in cancer cells changes nuclear architecture by affecting lamin expression, which downregulates genes maintaining cell-cell junctions resulting in increased cell migration.

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.

Sustained expression of FOXM1 is a hallmark of nearly all human cancers including squamous cell carcinomas of the head and neck (HNSCC). HNSCCs partially preserve the epithelial differentiation program, which recapitulates fetal and adult traits of the tissue of tumor origin but is deregulated by genetic alterations and tumor-supporting pathways. Using shRNA-mediated knockdown, we demonstrate a minimal impact of FOXM1 on proliferation and migration of HNSCC cell lines under standard cell culture conditions. However, FOXM1 knockdown in three-dimensional (3D) culture and xenograft tumor models resulted in reduced proliferation, decreased invasion, and a more differentiated-like phenotype, indicating a context-dependent modulation of FOXM1 activity in HNSCC cells. By ectopic overexpression of FOXM1 in HNSCC cell lines, we demonstrate a reduced expression of cutaneous-type keratin K1 and involucrin as a marker of squamous differentiation, supporting the role of FOXM1 in modulation of aberrant differentiation in HNSCC. Thus, our data provide a strong rationale for targeting FOXM1 in HNSCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keratin 15 (K15), a type I keratin, which pairs with K5 in epidermis, has been used extensively as a biomarker for stem cells. Two commercial antibodies, LHK15, a mouse monoclonal and EPR1614Y, a rabbit monoclonal, have been widely employed to study K15 expression. Here we report differential reactivity of these antibodies on epithelial cells and tissue sections. Although the two antibodies specifically recognised K15 on western blot, they reacted differently on skin sections and cell lines. LHK15 reacted in patches, whereas EPR1614Y reacted homogenously with the basal keratinocytes in skin sections. In cultured cells, LHK15 did not react with K15 deficient NEB-1, KEB-11, MCF-7 and SW13 cells expressing only exogenous K8 and K18 but reacted when these cells were transduced with K15. On the other hand, EPR1614Y reacted with these cells even though they were devoid of K15. Taken together these results suggest that EPR1614Y recognises a conformational epitope on keratin filaments which can be reconstituted by other keratins as well as by K15. In conclusion, this report highlights that all commercially available antibodies may not be equally specific in identifying the K15 positive stem cell.

Objective Besides angiotensin converting enzyme 2 (ACE2), an active involvement of proteases (FURIN and/or TMPRSS2) is important for cellular entry of SARS‐CoV‐2. Therefore, a simultaneous expression profiling of entry proteins in a tissue might provide a better risk assessment of SARS‐CoV‐2 infection as compared to individual proteins. In an attempt to understand the relative susceptibility of oral squamous cell carcinoma (OSCC) lesions as compared to the normal oral mucosa (NOM) for SARS‐CoV‐2 attachment/entry, this study examined the mRNA and protein expression profiles of ACE2, FURIN, and TMPRSS2 in the corresponding tissues using public transcriptomic and proteomics datasets. Methods and methods Public transcriptomic and proteomics datasets (the Cancer Genome Atlas (TCGA)/the Genotype‐Tissue Expression (GTEx), the Human Protein Atlas (HPA), and two independent microarray datasets) were used to examine the expression profiles of ACE2, TMPRSS2 and FURIN in NOM and OSCC. Results ACE2, TMPRSS2, and FURIN mRNAs were detected in NOM, however, at lower levels as compared to other body tissues. Except for moderate up‐regulation of FURIN, expression levels of ACE2 and TMPRSS2 mRNA were unchanged/down‐regulated in OSCC as compared to the NOM. Conclusions These results indicate that NOM may serve as a possible site for SARS‐CoV‐2 attachment, however, to a lesser extent as compared to organs with higher expression levels of the SARS‐CoV‐2 entry proteins. However, the evidence is lacking to suggest that expression status of entry proteins predisposes OSCC lesions to additional risk for SARS‐CoV‐2 attachment/entry as compared to NOM.

An interactive effect between nicotine and 3,4 methylenedioxymethamphetamine (MDMA) has been reported but the mechanism underlying such interaction is not completely understood. This study used zebrafish to explore gene expression changes associated with altered sensitivity to the rewarding effects of MDMA following 2 week exposure to nicotine and 2-60 days of nicotine withdrawal. Reward responses to MDMA were assessed using a conditioned place preference (CPP) paradigm and gene expression was evaluated using quantitative real time PCR of mRNA from whole brain samples from drug-treated and control adult zebrafish. Zebrafish pre-exposed for 2 weeks to nicotine showed increased conditioned place preference in response to low dose, 0.1 mg/kg, MDMA compared to un-exposed fish at 2, 7, 30 and 60 days withdrawal. Pre-exposure to nicotine for two weeks induced a significant increase of c-Fos and vasopressin receptor expression but a decrease of D(3) dopaminergic and oxytocin receptor expression at 2 days of withdrawal. C-Fos mRNA increased also at 7, 30, 60 days of withdrawal. Nicotine pre-exposed zebrafish submitted to MDMA-induced CPP, showed an increase in expression of p35 at day 2, α4 at day 30, vasopressin at day 7, and D(3) dopaminergic receptor at day 7, 30, and 60. These gene alterations could account for the altered sensitivity to the rewarding effects of MDMA in nicotine pre-exposed fish, suggesting that zebrafish have an altered ability to modulate behaviour as a function of reward during nicotine withdrawal.

Nasopharyngeal carcinoma (NPC) is a highly aggressive tumor characterized by distant metastasis. Deletion or down-regulation of the tumor suppressor protein ras-association domain family protein1 isoform A (RASSF1A) has been confirmed to be a key event in NPC progression; however, little is known about the effects or underlying mechanism of RASSF1A on the malignant phenotype. In the present study, we observed that RASSF1A expression inhibited the malignant phenotypes of NPC cells. Stable silencing of RASSF1A in NPC cell lines induced self-renewal properties and tumorigenicity in vivo/in vitro and the acquisition of an invasive phenotype in vitro. Mechanistically, RASSF1A inactivated Yes-associated Protein 1 (YAP1), a transcriptional coactivator, through actin remodeling, which further contributed to Platelet Derived Growth Factor Subunit B (PDGFB) transcription inhibition. Treatment with ectopic PDGFB partially increased the malignancy of NPC cells with transient knockdown of YAP1. Collectively, these findings suggest that RASSF1A inhibits malignant phenotypes by repressing PDGFB expression in a YAP1-dependent manner. PDGFB may serve as a potential interest of therapeutic regulators in patients with metastatic NPC.

BACKGROUND: Although there is clear evidence of genetic contributions to susceptibility to nicotine addiction, it has proved difficult to identify causal alleles and pathways from studies in humans. Mutagenesis in model species generates strong phenotypes not present in wildtype populations and can be used to identify biological mechanisms underlying quantifiable behaviours. We tested the hypothesis that a forward genetic screen for nicotine preference in zebrafish can predict loci and biological mechanisms influencing human smoking behaviour. METHODS: A population-based forward genetic screen of ethylnitrosurea- mutagenized zebrafish was used to identify lines of fish showing altered nicotine preference. Immunohistochemical, behavioral and quantitative PCR analyses were used to characterize mutant larvae. Focussed SNP analysis of the homologous human locus in cohorts from the UK and a Finnish Twin study assessed the predictive validity of the zebrafish data for human smoking behavior. RESULTS: We show nicotine preference is heritable in fish as in humans and identify loss-of-function mutations in the zebrafish Slit3 gene as associated with increased nicotine preference. Slit3 mutant larval fish showed altered sensitivity of habituation to acoustic startle to dopaminergic antagonists and increased Drd2 and Drd3 mRNA expression. Dopaminergic neuronal pathfinding was unaffected. Analysis of the SLIT3 locus in two independent human cohorts identified 2 genetic markers that predict level of cigarette consumption and likelihood of cessation. CONCLUSION: These findings suggest a role for SLIT3 signaling in development of dopaminergic pathways affecting behaviours associated with nicotine dependence and confirm the translational relevance of the zebrafish model in exploring complex human behaviors.

Desmoglein 3 (Dsg3) plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction in keratinocytes. A direct comparison was made with E-cadherin, a well-characterized mechanosensor. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in the expression and localization of junction assembly proteins. The knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of yes-associated protein (YAP), a mechanosensory, and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 formed a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to the strain or substrate rigidity-induced nuclear relocation of YAP and phospho-YAP. Plakophilin 1 (PKP1) seemed to be crucial in recruiting the complex containing Dsg3/phospho-YAP to the cell surface since its silencing affected Dsg3 junctional assembly with concomitant loss of phospho-YAP at the cell periphery. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes and cell proliferation. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

Desmoglein 3 (Dsg3), plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction. A direct comparison was made with E-cadherin, a well-characterized mechanosensor, in human keratinocytes. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in expression and localization of junction assembly proteins. Knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of YAP, a mechanosensor and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 forms a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to both strain-or substrate rigidity-induced nuclear relocalization of YAP and phospho-YAP. We showed that PKP1 seemed to be the key in such a complex formation since its silencing resulted in Dsg3 disruption at the junctions with concomitant loss of pYAP in the peripheral cytoplasm. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression ofYAP1target genes as well as cell proliferation in keratinocytes. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

Background Exosomes are extracellular vesicles released by almost all cell types, including cancer cells, into bodily fluids such as saliva, plasma, breast milk, semen, urine, cerebrospinal fluid, amniotic fluid, synovial fluid and sputum. Their key function being intercellular communication with both neighbouring as well as distant cells. Cancer exosomes have been shown to regulate organ-specific metastasis. However, little is known about the functional differences and molecular consequences of normal cells responding to exosomes derived from normal cells compared to those derived from cancer cells. Methods Here, we characterised and compared the transcriptome profiles of primary human normal oral keratinocytes (HNOK) in response to exosomes isolated from either primary HNOK or head and neck squamous cell carcinoma (HNSCC) cell lines. Results In recipient HNOK cells, we found that regardless of normal or cancer derived, exosomes altered molecular programmes involved in matrix modulation (MMP9), cytoskeletal remodelling (TUBB6, FEZ1, CCT6A), viral/dsRNA-induced interferon (OAS1, IFI6), anti-inflammatory (TSC22D3), deubiquitin (OTUD1), lipid metabolism and membrane trafficking (BBOX1, LRP11, RAB6A). Interestingly, cancer exosomes, but not normal exosomes, modulated expression of matrix remodelling (EFEMP1, DDK3, SPARC), cell cycle (EEF2K), membrane remodelling (LAMP2, SRPX), differentiation (SPRR2E), apoptosis (CTSC), transcription/translation (KLF6, PUS7). We have also identified CEP55 as a potential cancer exosomal marker. Conclusions In conclusion, both normal and cancer exosomes modulated unique gene expression pathways in normal recipient cells. Cancer cells may exploit exosomes to confer transcriptome reprogramming that leads to cancer-associated pathologies such as angiogenesis, immune evasion/modulation, cell fate alteration and metastasis. Molecular pathways and biomarkers identified in this study may be clinically exploitable for developing novel liquid-biopsy based diagnostics and immunotherapies. Electronic supplementary material The online version of this article (10.1186/s12943-018-0846-5) contains supplementary material, which is available to authorized users.

Background Nicotine withdrawal syndrome is a major clinical problem. Animal models with sufficient predictive validity to support translation of pre-clinical findings to clinical research are lacking. Aims We evaluated the behavioural and neurochemical alterations in zebrafish induced by short- and long-term nicotine withdrawal. Methods Zebrafish were exposed to 1 mg/L nicotine for 2 weeks. Dependence was determined using behavioural analysis following mecamylamine-induced withdrawal, and brain nicotinic receptor binding studies. Separate groups of nicotine-exposed and control fish were assessed for anxiety-like behaviours, anhedonia and memory deficits following 2–60 days spontaneous withdrawal. Gene expression analysis using whole brain samples from nicotine-treated and control fish was performed at 7 and 60 days after the last drug exposure. Tyrosine hydroxylase (TH) immunoreactivity in pretectum was also analysed. Results Mecamylamine-precipitated withdrawal nicotine-exposed fish showed increased anxiety-like behaviour as evidenced by increased freezing and decreased exploration. 3H-Epibatidine labeled heteromeric nicotinic acethylcholine receptors (nAChR) significantly increased after 2 weeks of nicotine exposure while 125I-αBungarotoxin labeled homomeric nAChR remained unchanged. Spontaneous nicotine withdrawal elicited anxiety-like behaviour (increased bottom dwelling), reduced motivation in terms of no preference for the enriched side in a place preference test starting from Day 7 after withdrawal and a progressive decrease of memory attention (lowering discrimination index). Behavioural differences were associated with brain gene expression changes: nicotine withdrawn animals showed decreased expression of chrna 4 and chrna7 after 60 days, and of htr2a from 7 to 60 days.The expression of c-Fos was significantly increased at 7 days. Finally, Tyrosine hydroxylase (TH) immunoreactivity increased in dorsal parvocellular pretectal nucleus, but not in periventricular nucleus of posterior tuberculum nor in optic tectum, at 60 days after withdrawal. Conclusions Our findings show that nicotine withdrawal induced anxiety-like behaviour, cognitive alterations, gene expression changes and increase in pretectal TH expression, similar to those observed in humans and rodent models.

Simple Summary Vimentin is an important filamentous protein providing structural and functional support to the cell. During initial stages of cancer development, vimentin concentration is very low, however, it increases when cancer starts to invade the surrounding areas. This review highlights the varied roles of vimentin in cancer growth and its spread to distant areas of the body. We have tried to explore the potential new areas of research related to the role of vimentin in cancer progression. We have also highlighted the reported damage to the vimentin gene in cancers, although how the damaged vimentin helps in cancer growth and spread is not known. We propose that latest technologies should be employed to medicinally target vimentin to reduce the cancer growth and its spread thereby helping to increase treatment outcomes and patients’ survival. Abstract Epithelial-mesenchymal transition (EMT) is a reversible plethora of molecular events where epithelial cells gain the phenotype of mesenchymal cells to invade the surrounding tissues. EMT is a physiological event during embryogenesis (type I) but also happens during fibrosis (type II) and cancer metastasis (type III). It is a multifaceted phenomenon governed by the activation of genes associated with cell migration, extracellular matrix degradation, DNA repair, and angiogenesis. The cancer cells employ EMT to acquire the ability to migrate, resist therapeutic agents and escape immunity. One of the key biomarkers of EMT is vimentin, a type III intermediate filament that is normally expressed in mesenchymal cells but is upregulated during cancer metastasis. This review highlights the pivotal role of vimentin in the key events during EMT and explains its role as a downstream as well as an upstream regulator in this highly complex process. This review also highlights the areas that require further research in exploring the role of vimentin in EMT. As a cytoskeletal protein, vimentin filaments support mechanical integrity of the migratory machinery, generation of directional force, focal adhesion modulation and extracellular attachment. As a viscoelastic scaffold, it gives stress-bearing ability and flexible support to the cell and its organelles. However, during EMT it modulates genes for EMT inducers such as Snail, Slug, Twist and ZEB1/2, as well as the key epigenetic factors. In addition, it suppresses cellular differentiation and upregulates their pluripotent potential by inducing genes associated with self-renewability, thus increasing the stemness of cancer stem cells, facilitating the tumour spread and making them more resistant to treatments. Several missense and frameshift mutations reported in vimentin in human cancers may also contribute towards the metastatic spread. Therefore, we propose that vimentin should be a therapeutic target using molecular technologies that will curb cancer growth and spread with reduced mortality and morbidity.

Background The concept of head and neck cancers (HNSCC) having unique molecular signatures is well accepted but relating this to clinical presentation and disease behaviour is essential for patient benefit. Currently the clinical significance of HNSCC molecular subtypes is uncertain therefore personalisation of HNSCC treatment is not yet possible. Methods We performed meta-analysis on 8 microarray studies and identified six significantly up- (PLAU, FN1, CDCA5) and down-regulated (CRNN, CLEC3B and DUOX1) genes which were subsequently quantified by RT-qPCR in 100 HNSCC patient margin and core tumour samples. Results Retrospective correlation with sociodemographic and clinicopathological patient details identified two subgroups of opposing molecular signature (+q6 and -q6) that correlated to two recognised high-risk HNSCC populations in the UK. The +q6 group were older, male, and excessive alcohol users whilst the –q6 group were younger, female, paan-chewers and predominantly Bangladeshi. Additionally, all patients with tumour recurrence were in the latter subgroup. Conclusions We provide the first evidence linking distinct molecular signatures in HNSCC with clinical presentations. Prospective trials are required to determine the correlation between these distinct genotypes and disease progression or treatment response. This is an important step towards the ultimate goal of improving outcomes by utilising personalised molecular-signature-guided treatments for HNSCC patients.

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell–cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3−/− mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/− controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell–cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signaling pathways implicated in the pathogenesis the PV blistering disease. We show here that expression of Dsg3 may directly influence p53, a key transcription factor governing the response to cellular stress. Dsg3 depletion caused increased p53 and apoptosis, an effect that was further enhanced by UV and mechanical strain and reversed by Dsg3 gain-of-function studies. Analysis in Dsg3-/- mouse skin confirmed increased p53/p21/caspase-3 compared to Dsg3+/- control in vivo. This Dsg3-p53 pathway involved YAP since Dsg3 forms a complex with YAP and regulates its expression and localization. Analysis of PV patient samples detected increased p53/YAP with diffuse cytoplasmic and/or nuclear staining in cells surrounding blisters. Treatment of keratinocytes with PV sera evoked pronounced p53/YAP expression. Collectively, our findings establish a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, suggesting that this pathway, involving YAP-Hippo control of skin homeostasis, is disrupted in PV.

Figure S1. RT-qPCR confirmed differential mRNA sorting into exosomes. Figure S2. Full length mRNA transcripts were found in exosomes and could be transferred into recipient cells. Figure S3. Exosomes induce morphological change within 24Â h in SVpgC2a recipient cells. (DOCX 981 kb)

Background Cancer-associated fibroblasts (CAFs) were shown to be important for tumour progression in head and neck squamous cell carcinomas (HNSCCs). Their heterogeneity and lack of specific markers is increasingly recognized. Integrin α11 was recently shown to be expressed by CAFs and might serve as a specific CAF marker. Aim To investigate integrin α11 expression and its correlation with the expression of a well-known marker of CAF, alpha smooth muscle actin (α-SMA), in HNSCC. Methods Fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples from healthy volunteers (n = 24), oral lichen planus (OLP) (n = 32) and HNSCC (n = 106) were collected together with clinical data after ethical approval. Immunohistochemistry to detect integrin α11 and α-SMA was performed on FF and FFPE samples. qPCR for integrin α11 (ITGA11) and α-SMA (ACTA2) was performed on FF samples. Data were analysed using chi-square test and Kaplan–Meier survival analysis. Results Significantly higher levels of integrin α11 and α-SMA at both protein and mRNA levels were found in HNSCC vs. normal controls and OLP. A strong correlation was found between integrin α11 and α-SMA expression, and double staining showed their colocalization. Both integrin α11 and α-SMA were detected surrounding metastatic islands. Expression of α-SMA at tumour front but not tumour centre correlated with patient survival. Conclusion Integrin α11 was overexpressed in HNSCC stroma and colocalized with α-SMA. Expression of α-SMA at tumour front but not tumour centre had prognostic value for survival, pinpointing the importance of assessing tumour front when evaluating stromal molecules as prognostic biomarkers.

Exposure to alcohol during early central nervous system development has been shown variously to affect aspects of physiological and behavioural development. In extreme cases, this can extend to craniofacial defects, severe developmental delay and mental retardation. At more moderate levels, subtle differences in brain morphology and behaviour have been observed. One clear effect of developmental alcohol exposure is an increase in the propensity to develop alcoholism and other addictions. The mechanisms by which this occurs, however, are not currently understood. In this study, we tested the hypothesis that adult zebrafish chronically exposed to moderate levels of ethanol during early brain ontogenesis would show an increase in conditioned place preference for alcohol and an increased propensity towards habit formation, a key component of drug addiction in humans. We found support for both of these hypotheses and found that the exposed fish had changes in mRNA expression patterns for dopamine receptor, nicotinic acetylcholine receptor and μ-opioid receptor encoding genes. Collectively, these data show an explicit link between the increased proclivity for addiction and addiction-related behaviour following exposure to ethanol during early brain development and alterations in the neural circuits underlying habit learning.

BACKGROUND Epigenetic reprogramming of the methylome has been implicated in all stages of cancer evolution. It is now well accepted that cancer cells exploit epigenetic reprogramming, a mechanism that regulates stem/progenitor cell renewal and differentiation, to promote cancer initiation and progression. The oncogene FOXM1 has been implicated in all major forms of human cancer. METHODS We have recently shown that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a number of FOXM1-induced epigenetic markers. Differential promoter methylation and gene expression in clinical specimens were measured using commercially available bisulfite conversion kits and absolute quantitative PCR, respectively. RESULTS Here, we investigated 8 FOXM1-induced differentially methylated genes, SPCS1, FLNA, CHPF, GLT8D1, C6orf136, MGAT1, NDUFA10, and PAFAH1B3, using human head and neck tissue specimens donated by 2 geographically independent patient cohorts from Norway and the United Kingdom. Two genes (GLT8D1 and C6orf136) were found to be differentially expressed in head and neck squamous cell carcinomas (HNSCCs). Using methylation-specific quantitative PCR, we confirmed that the promoters of GLT8D1 and C6orf136 were hypo- and hypermethylated, respectively, in HNSCC tissues. CONCLUSIONS Given that epigenetic change precedes gene expression, methylation status of candidate genes identified from this study may represent a signature of premalignancy, rendering them potentially useful predictive biomarkers for precancer screening and/or therapeutic targets for cancer prevention. Cancer 2013;119:4249–4258. © 2013 American Cancer Society.

Keratin 15 (K15) is type I keratin protein co-expressed with the K5/K14 pair present in the basal keratinocytes of all stratified epithelia. Although it is a minor component of the cytoskeleton with a variable expression pattern, nonetheless its expression has been reported as a stem cell marker in the bulge of hair follicles. Conversely, suprabasal expression of K15 has also been reported in both normal and diseased tissues, which is inconsistent with its role as a stem cell marker. Our recently published work has given evidence of the molecular pathways that seem to control the expression of K15 in undifferentiated and differentiated cells. In this article, we have critically reviewed the published work to establish the reliability of K15 as an epidermal stem cell marker.

The forkhead box M1 (FOXM1) transcription factor gene has been implicated in almost all human cancer types. It would be an ideal biomarker for cancer detection but, to date, its translation into a cancer diagnostic tool is yet to materialise. The quantitative Malignancy Index Diagnostic System (qMIDS) was the first FOXM1 oncogene-based diagnostic test developed for quantifying squamous cell carcinoma aggressiveness. The test was originally validated using head and neck squamous cell carcinomas (HNSCC) from European patients. The HNSCC gene expression signature across geographical and ethnic differences is unknown. This is the first study evaluated the FOXM1-based qMIDS test using HNSCC specimens donated by ethnic Chinese patients. We tested 50 Chinese HNSCC patients and 18 healthy subjects donated 68 tissues in total. qMIDS scores from the Chinese cohort were compared with the European datasets (n = 228). The median ± SD scores for the Chinese cohort were 1.13 ± 0.66, 4.02 ± 1.66 and 5.83 ± 3.13 in healthy oral tissues, adjacent tumour margin and HNSCC core tissue, respectively. Diagnostic test efficiency between the Chinese and European datasets was almost identical. Consistent with previous European data, qMIDS scores for HNSCC samples were not influenced by gender or age. The degree of HNSCC differentiation, clinical stage and lymphatic metastasis status were found to be correlated with qMIDS scores. This study provided the first evidence that the pathophysiology of HNSCC was molecularly indistinguishable between the Chinese and European specimens. The qMIDS test robustly quantifies a universal FOXM1-driven oncogenic program, at least in HNSCC, which transcends ethnicity, age, gender and geographic origins.

Oral submucous fibrosis (OSMF) is associated with paan chewing, altered collagen metabolism, inflammation and the upregulation of numerous cytokines. OSMF fibroblasts accumulate senescent cells at an increased rate because of increased reactive oxygen species production and DNA double-strand breaks (DDBs), generated intrinsically by damaged mitochondria. This results in a reduced replicative lifespan. However, it is still unclear which other changes are intrinsic to the fibroblasts and associated with OSMF rather than the paan chewing habit or the OSMF environment. Both the oral epithelium and the mesenchyme have elevated levels of TGF-β(1) in OSMF in vivo. However, in cultured fibroblasts, secreted levels of TGF-β(1,) other cytokines and the matrix metalloproteinases 1 and 2 showed no association with OSMF. In contrast, the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, were increased in 10/11 OSMF fibroblast cultures relative to normal and non-diseased paan user controls. OSMF fibroblast collagen levels were normal. TIMP levels correlated with replicative lifespan of the cultures but not with the presence of senescent cells, as senescent cell depletion in OSMF fibroblast cultures did not result in a reduction in either TIMP-1 or TIMP-2. However, the introduction of unrepairable DDBs into normal oral fibroblasts by ionizing radiation increased TIMP-1 and TIMP-2 secretion by two-fold and seven-fold, respectively, within 5 days, replicating early senescence and the elevation seen in OSMF cultures. Therefore, increased fibroblast TIMP-1/2 levels could be early disease-specific markers of OSMF onset, DDBs and ageing and may have clinical significance, as OSMF can be reversed in its early stages.

Stem cells are permanent residents of tissues and thought to be targets of cancer initiation. The frequent, and often early, upregulation of the FOXM1 transcription factor in the majority of human cancers suggests that it may participate in the initiation of human tumorigenesis. However, this hypothesis has not been tested. Herein, we show that targeting the ectopic expression of FOXM1 to the highly clonogenic cells of primary human keratinocytes with stem/progenitor cell properties, but not to differentiating cells, caused clonal expansion in vitro. We show, using a functional three-dimensional organotypic epithelial tissue regeneration system, that ectopic FOXM1 expression perturbed epithelial differentiation generating a hyperproliferative phenotype reminiscent of that seen in human epithelial hyperplasia. Furthermore, transcriptional expression analysis of a panel of 28 epithelial differentiation-specific genes reveals a role for FOXM1 in the suppression of epithelial differentiation. This study provides the first evidence that FOXM1 participates in an early oncogenic pathway that predisposes cells to tumorigenesis by expanding the stem/progenitor compartment and deregulating subsequent keratinocyte terminal differentiation. This finding reveals an important window of susceptibility to oncogenic signals in epithelial stem/progenitor cells prior to differentiation, and may provide a significant benefit to the design of cancer therapeutic interventions that target oncogenesis at its earliest incipient stage. Cancer Res; 70(22); 9515–26. ©2010 AACR.

Desmosomes, the intercellular junctions that confer strong adhesion between epithelial cells, are frequently altered in malignancy. However, a comprehensive analysis of these structures has not been carried out in oral neoplasia. Oral squamous cell carcinomas (SCCs) and pre-malignant dysplasia can be sub-classified according to their in vitro replicative lifespan, where the immortal dysplasia (ID) and carcinoma (IC) subsets have p16(ink4a) and p53 dysfunction, telomerase deregulation and genetic instability and the mortal subset (MD and MC) do not. We found that the desmosomal proteins exhibit a distinct expression pattern in oral mucosa when compared with epidermis in vivo. Microarray data from a large panel of lines revealed that the transcript levels of DSG3, DSC2/3, DP, PG and PKP1 were reduced in ID and IC. Interestingly, DSG2 was up-regulated in MC. Reduction of DSG3 and up-regulation of DSG2 were found in two independent microarray datasets. Significantly, we demonstrated that reduction of DSG3 and up-regulation of DSG2 was reversible in vitro by using RNAi-mediated knockdown of DSG2 in IC cells. The remaining desmosomal proteins were largely disrupted or internalized and associated with retraction of keratin intermediate filaments in oral SCC lines. These findings suggest dysfunction and loss of desmosomal components are common events in the immortal class of oral SCC and that these events may precede overt malignancy.