A Review of the Repair of DNA Double Strand Breaks in the Development of Oral Cancer.

We explore the possibility that defects in genes associated with the response and repair of DNA double strand breaks predispose oral potentially malignant disorders (OPMD) to undergo malignant transformation to oral squamous cell carcinoma (OSCC). Defects in the homologous recombination/Fanconi anemia (HR/FA), but not in the non-homologous end joining, causes the DNA repair pathway to appear to be consistent with features of familial conditions that are predisposed to OSCC (FA, Bloom's syndrome, Ataxia Telangiectasia); this is true for OSCC that occurs in young patients, sometimes with little/no exposure to classical risk factors. Even in Dyskeratosis Congenita, a disorder of the telomerase complex that is also predisposed to OSCC, attempts at maintaining telomere length involve a pathway with shared HR genes. Defects in the HR/FA pathway therefore appear to be pivotal in conditions that are predisposed to OSCC. There is also some evidence that abnormalities in the HR/FA pathway are associated with malignant transformation of sporadic cases OPMD and OSCC. We provide data showing overexpression of HR/FA genes in a cell-cycle-dependent manner in a series of OPMD-derived immortal keratinocyte cell lines compared to their mortal counterparts. The observations in this study argue strongly for an important role of the HA/FA DNA repair pathway in the development of OSCC.

Multiproxy bioarchaeological data reveals interplay between growth, diet and population dynamics across the transition to farming in the central Mediterranean.

The transition to farming brought on a series of important changes in human society, lifestyle, diet and health. The human bioarchaeology of the agricultural transition has received much attention, however, relatively few studies have directly tested the interrelationship between individual lifestyle factors and their implications for understanding life history changes among the first farmers. We investigate the interplay between skeletal growth, diet, physical activity and population size across 30,000 years in the central Mediterranean through a 'big data' cross-analysis of osteological data related to stature (n = 361), body mass (n = 334) and long bone biomechanics (n = 481), carbon (δ13C) and nitrogen (δ15N) stable isotopes (n = 1986 human, n = 475 animal) and radiocarbon dates (n = 5263). We present the observed trends on a continuous timescale in order to avoid grouping our data into assigned 'time periods', thus achieving greater resolution and chronological control over our analysis. The results identify important changes in human life history strategies associated with the first farmers, but also highlight the long-term nature of these trends in the millennia either side of the agricultural transition. The integration of these different data is an important step towards disentangling the complex relationship between demography, diet and health, and reconstruct life history changes within a southern European context. We believe the methodological approach adopted here has broader global implications for bioarchaeological studies of human adaptation more generally.

Patient ratings in exercise therapy for the management of tendinopathy: a systematic review with meta-analysis.

OBJECTIVE: To synthesise exercise therapy intervention data investigating patient rating outcomes for the management of tendinopathy. DESIGN: A systematic review and meta-analysis of randomized controlled trials investigating exercise therapy interventions and reporting patient rating outcomes. SETTING: Any setting in any country listed as very high on the human development index. PARTICIPANTS: People with a diagnosis of any tendinopathy of any severity or duration. INTERVENTIONS: Exercise therapy for the management of tendinopathy comprising five different therapy classes: 1) resistance; 2) plyometric; 3) vibration; 4) flexibility, and 5) movement pattern retraining modalities, were considered for inclusion. MAIN OUTCOME MEASURES: Outcomes measuring patient rating of condition, including patient satisfaction and Global Rating of Change (GROC). RESULTS: From a total of 124 exercise therapy studies, 34 (Achilles: 41%, rotator cuff: 32%, patellar: 15%, elbow: 9% and gluteal: 3%) provided sufficient information to be meta-analysed. The data were obtained across 48 treatment arms and 1246 participants. The pooled estimate for proportion of satisfaction was 0.63 [95%CrI: 0.53-0.73], and the pooled estimate for percentage of maximum GROC was 53 [95%CrI: 38-69%]. The proportion of patients reporting positive satisfaction and perception of change increased with longer follow-up periods from treatment onset. CONCLUSION: Patient satisfaction and GROC appear similar and are ranked moderately high demonstrating that patients generally perceive exercise therapies positively. Further research including greater consistency in measurement tools is required to explore and where possible, identify patient- and exercise-related moderating factors that can be used to improve person-centred care. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO ID=CRD42020168187 CONTRIBUTION OF PAPER.

Escape from Cellular Senescence Is Associated with Chromosomal Instability in Oral Pre-Malignancy.

An escape from cellular senescence through the development of unlimited growth potential is one of the hallmarks of cancer, which is thought to be an early event in carcinogenesis. In this review, we propose that the molecular effectors of senescence, particularly the inactivation of TP53 and CDKN2A, together with telomere attrition and telomerase activation, all lead to aneuploidy in the keratinocytes from oral potentially malignant disorders (OPMD). Premalignant keratinocytes, therefore, not only become immortal but also develop genotypic and phenotypic cellular diversity. As a result of these changes, certain clonal cell populations likely gain the capacity to invade the underlying connective tissue. We review the clinical implications of these changes and highlight a new PCR-based assay to identify aneuploid cell in fluids such as saliva, a technique that is extremely sensitive and could facilitate the regular monitoring of OPMD without the need for surgical biopsies and may avoid potential biopsy sampling errors. We also draw attention to recent studies designed to eliminate aneuploid tumour cell populations that, potentially, is a new therapeutic approach to prevent malignant transformations in OPMD.

Extracellular citrate and metabolic adaptations of cancer cells.

It is well established that cancer cells acquire energy via the Warburg effect and oxidative phosphorylation. Citrate is considered to play a crucial role in cancer metabolism by virtue of its production in the reverse Krebs cycle from glutamine. Here, we review the evidence that extracellular citrate is one of the key metabolites of the metabolic pathways present in cancer cells. We review the different mechanisms by which pathways involved in keeping redox balance respond to the need of intracellular citrate synthesis under different extracellular metabolic conditions. In this context, we further discuss the hypothesis that extracellular citrate plays a role in switching between oxidative phosphorylation and the Warburg effect while citrate uptake enhances metastatic activities and therapy resistance. We also present the possibility that organs rich in citrate such as the liver, brain and bones might form a perfect niche for the secondary tumour growth and improve survival of colonising cancer cells. Consistently, metabolic support provided by cancer-associated and senescent cells is also discussed. Finally, we highlight evidence on the role of citrate on immune cells and its potential to modulate the biological functions of pro- and anti-tumour immune cells in the tumour microenvironment. Collectively, we review intriguing evidence supporting the potential role of extracellular citrate in the regulation of the overall cancer metabolism and metastatic activity.

Targeting the genetic landscape of oral potentially malignant disorders has the potential as a preventative strategy in oral cancer.

This study reviews the molecular landscape of oral potentially malignant disorders (OPMD). We examine the impact of tumour heterogeneity, the spectrum of driver mutations (TP53, CDKN2A, TERT, NOTCH1, AJUBA, PIK3CA, CASP8) and gene transcription on tumour progression. We comment on how some of these mutations impact cellular senescence, field cancerization and cancer stem cells. We propose that OPMD can be monitored more closely and more dynamically through the use of liquid biopsies using an appropriate biomarker of transformation. We describe new gene interactions through the use of a systems biology approach and we highlight some of the first studies to identify functional genes using CRISPR-Cas9 technology. We believe that this information has translational implications for the use of re-purposed existing drugs and/or new drug development. Further, we argue that the use of digital technology encompassing clinical and laboratory-based data will create relevant datasets for machine learning/artificial intelligence. We believe that therapeutic intervention at an early molecular premalignant stage should be an important preventative strategy to inhibit the development of oral squamous cell carcinoma and that this approach is applicable to other aerodigestive tract cancers.

Autophagy is deregulated in cancer-associated fibroblasts from oral cancer and is stimulated during the induction of fibroblast senescence by TGF-ß1.

Many of the characteristics ascribed to cancer-associated fibroblasts (CAFs) are shared by activated, autophagic and senescent fibroblasts. Whilst most oral squamous cell carcinomas (OSCCs) are genetically unstable (GU-OSCC), genetically stable variants (GS-OSCC) have been described and, notably, CAF activation (myofibroblast differentiation) and senescence are characteristics particularly associated with GU-OSCCs. However, it is not known whether autophagy is disrupted in these cells or whether autophagy regulates the development of the myofibroblast and senescent phenotypes. In this study, we show that senescent CAFs from GU-OSCCs contained more autophagosomes than normal human oral fibroblasts (NHOFs) and CAFs from GS-OSCCs possibly due to autophagic impairment. Further, we show that deregulation of autophagy in normal fibroblasts, either by inhibition with autophagy inhibitor, SAR405, or activation with TGF-ß1, induced fibroblast activation and senescence: In response to TGF-ß1, autophagy was induced prior to the development of the activated and senescent phenotypes. Lastly, we show that both SAR405- and TGF-ß1-treated NHOFs enhance OSCC cell migration but only TGF-ß1-treated cells increase OSCC invasion through Matrigel, indicating that TGF-ß1 has additional effects that are independent of fibroblast activation/senescence. These results suggest a functional role for autophagy in the development of myofibroblast and CAF phenotypes.

Identification of proteins associated with development of metastasis from cutaneous squamous cell carcinomas (cSCCs) via proteomic analysis of primary cSCCs.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers capable of metastasizing. Proteomic analysis of cSCCs can provide insight into the biological processes responsible for metastasis, as well as future therapeutic targets and prognostic biomarkers. OBJECTIVES: To identify proteins associated with development of metastasis in cSCC. METHODS: A proteomic-based approach was employed on 105 completely excised, primary cSCCs, comprising 52 that had metastasized (P-M) and 53 that had not metastasized at 5 years post-surgery (P-NM). Formalin-fixed, paraffin-embedded cSCCs were microdissected and subjected to proteomic profiling after one-dimensional (1D), and separately two-dimensional (2D), liquid chromatography fractionation. RESULTS: A discovery set of 24 P-Ms and 24 P-NMs showed 144 significantly differentially expressed proteins, including 33 proteins identified via both 1D and 2D separation, between P-Ms and P-NMs. Several differentially expressed proteins were also associated with survival in SCCs of other organs. The findings were verified by multiple reaction monitoring on six peptides from two proteins, annexin A5 (ANXA5) and dolichyl-diphosphooligosaccharide-protein glycosyltransferase noncatalytic subunit (DDOST), in the discovery group and validated on a separate cohort (n = 57). Increased expression of ANXA5 and DDOST was associated with reduced time to metastasis in cSCC and decreased survival in cervical and oropharyngeal cancer. A prediction model using ANXA5 and DDOST had an area under the curve of 0·93 (confidence interval 0·83-1·00), an accuracy of 91·2% and higher sensitivity and specificity than cSCC staging systems currently in clinical use. CONCLUSIONS: This study highlights that increased expression of two proteins, ANXA5 and DDOST, is significantly associated with poorer clinical outcomes in cSCC.

Extracellular Citrate Fuels Cancer Cell Metabolism and Growth.

Cancer cells need excess energy and essential nutrients/metabolites not only to divide and proliferate but also to migrate and invade distant organs for metastasis. Fatty acid and cholesterol synthesis, considered a hallmark of cancer for anabolism and membrane biogenesis, requires citrate. We review here potential pathways in which citrate is synthesized and/or supplied to cancer cells and the impact of extracellular citrate on cancer cell metabolism and growth. Cancer cells employ different mechanisms to support mitochondrial activity and citrate synthesis when some of the necessary substrates are missing in the extracellular space. We also discuss the different transport mechanisms available for the entry of extracellular citrate into cancer cells and how citrate as a master metabolite enhances ATP production and fuels anabolic pathways. The available literature suggests that cancer cells show an increased metabolic flexibility with which they tackle changing environmental conditions, a phenomenon crucial for cancer cell proliferation and metastasis.

Collagen Induces a More Proliferative, Migratory and Chemoresistant Phenotype in Head and Neck Cancer via DDR1.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and includes squamous cell carcinomas of the oropharynx and oral cavity. Patient prognosis has remained poor for decades and molecular targeted therapies are not in routine use. Here we showed that the overall expression of collagen subunit genes was higher in cancer-associated fibroblasts (CAFs) than normal fibroblasts. Focusing on collagen8A1 and collagen11A1, we showed that collagen is produced by both CAFs and tumour cells, indicating that HNSCCs are collagen-rich environments. We then focused on discoidin domain receptor 1 (DDR1), a collagen-activated receptor tyrosine kinase, and showed that it is over-expressed in HNSCC tissues. Further, we demonstrated that collagen promoted the proliferation and migration of HNSCC cells and attenuated the apoptotic response to cisplatin. Knockdown of DDR1 in HNSCC cells demonstrated that these tumour-promoting effects of collagen are mediated by DDR1. Our data suggest that specific inhibitors of DDR1 might provide novel therapeutic opportunities to treat HNSCC.

The induction of the fibroblast extracellular senescence metabolome is a dynamic process.

Cellular senescence is often associated with irreparable DNA double strand breaks (IrrDSBs) which accumulate with chronological age (IrrDSBsen). The removal of senescent cells ameliorates several age-related diseases in mice but the translation of these findings into a clinical setting would be aided by the characterisation of non-invasive biomarkers of senescent cells. Several serum metabolites are independent indicators of chronological age and some of these accumulate outside senescent fibroblasts independently of cell cycle arrest, repairable DNA breaks and cell size (the extracellular senescence metabolome, or ESM). The post-mitotic phase of senescence is dynamic, making the detection of senescent cells in vivo difficult. An unbiased metabolomic screen of the IrrDSBsen fibroblast ESM also showed differences in the times of initiation and maintenance of different metabolites but generally the ESM altered progressively over the 20 day study period unlike the reported transcriptional profiles. This more detailed analysis of IrrDSBsen identified several new ESM metabolites that are associated with chronological ageing. Targeted analysis of citrate confirmed the dynamic nature of this metabolite in two cell lines and revealed its independence from the senescence effector p16INK4A. These data will aid our understanding of metabolic signatures of ageing and their relationship to cellular senescence and IrrDSBs.

A miRNA-145/TGF-ß1 negative feedback loop regulates the cancer-associated fibroblast phenotype.

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-ß1 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-ß1 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-ß signalling pathway. miR-145 was also overexpressed in CAF derived from oral cancers. Overexpression of miR-145 blocked TGF-ß1-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.

Perspectives and attitudes of pediatricians concerning post-discharge care practice of premature infants.

OBJECTIVE: Survival rates of premature infants are at a historical high and increasingly more pediatricians are caring for former premature infants. The goal of this study was to describe the perspectives and attitudes of pediatricians, as well as, the challenges of rendering post-neonatal intensive care unit (NICU) discharge care for premature infants. METHODS: An anonymous 22-question web-based survey was emailed to pediatricians who are current members of the American Academy of Pediatrics (AAP) and practicing in Kings County, New York. RESULTS: There were 148 completed surveys with 79% being general pediatricians. Of all respondents, 63% believed that premature infants should have a neonatal high risk follow-up visit within days after discharge and 64% were satisfied with the NICU discharge summary acquisition. While 74% of pediatricians felt comfortable following up with former extremely premature infants, 65% referred to specialists, most often to child development, neurology, and physical and/or occupational therapy. The majority (85%) were more likely to refer premature infants to early intervention. Participating pediatricians varied in their knowledge of immunization and breastfeeding guidelines. Finally, 88% of respondents acknowledged that caregivers of premature infants experience increased stress, with 53% stating that the stress should be addressed. CONCLUSIONS: Understanding the perceptions and challenges of pediatricians who care for premature infants may help improve post NICU quality of care. Transition to the outpatient setting is a crucial step in the management of premature infants and a focus on improved hand-off procedures between hospital and physicians may prove beneficial. Also, pediatricians must stay abreast of current recommendations for breastfeeding and vaccinations. Furthermore, emphasis should be given to stress reduction and management for caregivers of former premature infants.

Cancer-associated fibroblasts regulate keratinocyte cell-cell adhesion via TGF-ß-dependent pathways in genotype-specific oral cancer.

The interrelationship between malignant epithelium and the underlying stroma is of fundamental importance in tumour development and progression. In the present study, we used cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), tumours that are characterized by the loss of genes such as TP53 and p16INK4A and with extensive loss of heterozygosity, together with CAFs from their more genetically stable (GS) counterparts that have wild-type TP53 and p16INK4A and minimal loss of heterozygosity (GS-OSCC). Using a systems biology approach to interpret the genome-wide transcriptional profile of the CAFs, we show that transforming growth factor-ß (TGF-ß) family members not only had biological relevance in silico but also distinguished GU-OSCC-derived CAFs from GS-OSCC CAFs and fibroblasts from normal oral mucosa. In view of the close association between TGF-ß family members, we examined the expression of TGF-ß1 and TGF-ß2 in the different fibroblast subtypes and showed increased levels of active TGF-ß1 and TGF-ß2 in CAFs from GU-OSCC. CAFs from GU-OSCC, but not GS-OSCC or normal fibroblasts, induced epithelial-mesenchymal transition and down-regulated a broad spectrum of cell adhesion molecules resulting in epithelial dis-cohesion and invasion of target keratinocytes in vitro in a TGF-ß-dependent manner. The results demonstrate that the TGF-ß family of cytokines secreted by CAFs derived from genotype-specific oral cancer (GU-OSCC) promote, at least in part, the malignant phenotype by weakening intercellular epithelial adhesion.

Fibroblast activation and senescence in oral cancer.

There is now compelling evidence that the tumour stroma plays an important role in the pathogenesis of cancers of epithelial origin. The pre-eminent cell type of the stroma is carcinoma-associated fibroblasts. These cells demonstrate remarkable heterogeneity with activation and senescence being common stress responses. In this review, we summarise the part that these cells play in cancer, particularly oral cancer, and present evidence to show that activation and senescence reflect a unified programme of fibroblast differentiation. We report advances concerning the senescent fibroblast metabolome, mechanisms of gene regulation in these cells and ways in which epithelial cell adhesion is dysregulated by the fibroblast secretome. We suggest that the identification of fibroblast stress responses may be a valuable diagnostic tool in the determination of tumour behaviour and patient outcome. Further, the fact that stromal fibroblasts are a genetically stable diploid cell population suggests that they may be ideal therapeutic targets and early work in this context is encouraging.

Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism.

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.

Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner.

BACKGROUND: Limited options for the treatment of cartilage damage have driven the development of tissue engineered or cell therapy alternatives reliant on ex vivo cell expansion. The study of chondrogenesis in primary cells is difficult due to progressive cellular aging and senescence. Immortalisation via the reintroduction of the catalytic component of telomerase, hTERT, could allow repeated, longitudinal studies to be performed while bypassing senescent phenotypes. METHODS: Three human cell types: bone marrow-derived stromal cells (BMA13), embryonic stem cell-derived (1C6) and chondrocytes (OK3) were transduced with hTERT (BMA13H, 1C6H and OK3H) and proliferation, surface marker expression and tri-lineage differentiation capacity determined. The sulphated glycosaminoglycan (sGAG) content of the monolayer and spent media was quantified in maintenance media (MM) and pro-chondrogenic media (PChM) and normalised to DNA. RESULTS: hTERT expression was confirmed in transduced cells with proliferation enhancement in 1C6H and OK3H cells but not BMA13H. All cells were negative for leukocyte markers (CD19, CD34, CD45) and CD73 positive. CD14 was expressed at low levels on OK3 and OK3H and HLA-DR on BMA13 (84.8%). CD90 was high for BMA13 (84.9%) and OK3 (97.3%) and moderate for 1C6 (56.7%), expression was reduced in BMA13H (33.7%) and 1C6H (1.6%). CD105 levels varied (BMA13 87.7%, 1C6 8.2%, OK3 43.3%) and underwent reduction in OK3H (25.1%). 1C6 and BMA13 demonstrated osteogenic and adipogenic differentiation but mineralised matrix and lipid accumulation appeared reduced post hTERT transduction. Chondrogenic differentiation resulted in increased monolayer-associated sGAG in all primary cells and 1C6H (p<0.001), and BMA13H (p<0.05). In contrast OK3H demonstrated reduced monolayer-associated sGAG in PChM (p<0.001). Media-associated sGAG accounted for ≥55% (PChM-1C6) and ≥74% (MM-1C6H). CONCLUSION: In conclusion, hTERT transduction could, but did not always, prevent senescence and cell phenotype, including differentiation potential, was affected in a variable manner. As such, these cells are not a direct substitute for primary cells in cartilage regeneration research.

Senescent cancer-associated fibroblasts secrete active MMP-2 that promotes keratinocyte dis-cohesion and invasion.

BACKGROUND: Previous studies have demonstrated that senescent cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), unlike non-senescent CAFs from genetically stable carcinomas (GS-OSCC), promoted keratinocyte invasion in vitro in a paracrine manner. The mechanism by which this occurs is unclear. METHODS: Previous work to characterise the senescent-associated secretory phenotype (SASP) has used antibody arrays, technology that is limited by the availability of suitable antibodies. To extend this work in an unbiased manner, we used 2D gel electrophoresis and mass spectroscopy for protein identification. Matrix metalloproteinases (MMPs) were investigated by gelatin zymography and western blotting. Neutralising antibodies were used to block key molecules in the functional assays of keratinocyte adhesion and invasion. RESULTS: Among a variety of proteins that were differentially expressed between CAFs from GU-OSCC and GS-OSCC, MMP-2 was a major constituent of senescent CAF-CM derived from GU-OSCC. The presence of active MMP-2 was confirmed by gelatine zymography. MMP-2 derived from senescent CAF-CM induced keratinocyte dis-cohesion and epithelial invasion into collagen gels in a TGF-ß-dependent manner. CONCLUSIONS: Senescent CAFs from GU-OSCC promote a more aggressive oral cancer phenotype by production of active MMP-2, disruption of epithelial adhesion and induction of keratinocyte invasion.