Peripheral (not central) corneal epithelia contribute to the closure of an annular debridement injury.

Corneal epithelia have limited self-renewal and therefore reparative capacity. They are continuously replaced by transient amplifying cells which spawn from stem cells and migrate from the periphery. Because this view has recently been challenged, our goal was to resolve the conflict by giving mice annular injuries in different locations within the corneolimbal epithelium, then spatiotemporally fate-mapping cell behavior during healing. Under these conditions, elevated proliferation was observed in the periphery but not the center, and wounds predominantly resolved by centripetally migrating limbal epithelia. After wound closure, the central corneal epithelium was completely replaced by K14+ limbal-derived clones, an observation supported by high-resolution fluorescence imaging of genetically marked cells in organ-cultured corneas and via computational modeling. These results solidify the essential role of K14+ limbal epithelial stem cells for wound healing and refute the notion that stem cells exist within the central cornea and that their progeny have the capacity to migrate centrifugally.

A Wnt-mediated phenotype switch along the epithelial-mesenchymal axis defines resistance and invasion downstream of ionising radiation in oral squamous cell carcinoma.

BACKGROUND: Dynamic transitions of tumour cells along the epithelial-mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance. METHODS: In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation. RESULTS: Our study demonstrates a prominent hybrid epithelial-mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial-mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion. CONCLUSION: This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.

Nicotinamide for skin cancer chemoprevention: effects of nicotinamide on melanoma in vitro and in vivo.

Nicotinamide (NAM), an amide form of vitamin B3, replenishes cellular energy after ultraviolet radiation (UVR) exposure, thereby enhancing DNA repair and reducing UVR's immunosuppressive effects. NAM reduces actinic keratoses and new keratinocyte cancers in high risk individuals, but its effects on melanoma are unknown. Melanomas arising on NAM or placebo within the ONTRAC skin cancer chemoprevention trial (Oral Nicotinamide To Reduce Actinic Cancer) were examined by immunohistochemistry. The effects of NAM (50 µM, 5 mM and 20 mM) on the viability, proliferation and invasiveness of four human melanoma cell lines and on the viability and proliferation of two human melanocyte lines, with and without UV irradiation were also investigated. 50 µM NAM did not affect viability, proliferation or invasion of melanoma or melanocyte cell lines, whereas concentrations too high to be achievable in vivo reduced viability and proliferation. Nicotinamide did not enhance melanoma viability, proliferation or invasiveness in vitro, providing additional confidence in its safety for use in clinical trials in high risk patients. Peritumoral and tumour infiltrating CD4+ and CD8+ lymphocytes were significantly increased in melanomas arising on NAM compared to those arising on placebo. Given the chemopreventive activity of nicotinamide against keratinocyte cancers, its DNA repair enhancing effects in melanocytes and now its potential enhancement of tumour-infiltrating lymphocytes and lack of adverse effects on melanoma cell growth and proliferation, clinical trials of nicotinamide for melanoma chemoprevention are now indicated.

α3 Integrin of Cell-Cell Contact Mediates Kidney Fibrosis by Integrin-Linked Kinase in Proximal Tubular E-Cadherin Deficient Mice.

Loss of E-cadherin marks a defect in epithelial integrity and polarity during tissue injury and fibrosis. Whether loss of E-cadherin plays a causal role in fibrosis is uncertain. α3ß1 Integrin has been identified to complex with E-cadherin in cell-cell adhesion, but little is known about the details of their cross talk. Herein, E-cadherin gene (Cdh1) was selectively deleted from proximal tubules of murine kidney by Sglt2Cre. Ablation of E-cadherin up-regulated α3ß1 integrin at cell-cell adhesion. E-cadherin-deficient proximal tubular epithelial cell displayed enhanced transforming growth factor-ß1-induced α-smooth muscle actin (α-SMA) and vimentin expression, which was suppressed by siRNA silencing of α3 integrin, but not ß1 integrin. Up-regulation of transforming growth factor-ß1-induced α-SMA was mediated by an α3 integrin-dependent increase in integrin-linked kinase (ILK). Src phosphorylation of ß-catenin and consequent p-ß-catenin-Y654/p-Smad2 transcriptional complex underlies the transcriptional up-regulation of ILK. Kidney fibrosis after unilateral ureteric obstruction or ischemia reperfusion was increased in proximal tubule E-cadherin-deficient mice in comparison to that of E-cadherin intact control mice. The exacerbation of fibrosis was explained by the α3 integrin-dependent increase of ILK, ß-catenin nuclear translocation, and α-SMA/proximal tubular-specific Cre double positive staining in proximal tubular epithelial cell. These studies delineate a nonconventional integrin/ILK signaling by α3 integrin-dependent Src/p-ß-catenin-Y654/p-Smad2-mediated up-regulation of ILK through which loss of E-cadherin leads to kidney fibrosis.

Bioengineered human heparin with anticoagulant activity.

Heparin is a carbohydrate anticoagulant used clinically to prevent thrombosis, however impurities can limit its efficacy. Here we report the biosynthesis of heparin-like heparan sulfate via the recombinant expression of human serglycin in human cells. The expressed serglycin was also decorated with chondroitin/dermatan sulfate chains and the relative abundance of these glycosaminoglycan chains changed under different concentrations of glucose in the culture medium. The recombinantly expressed serglycin produced with 25mM glucose present in the culture medium was found to possess anticoagulant activity one-seventh of that of porcine unfractionated heparin, demonstrating that bioengineered human heparin-like heparan sulfate may be a safe next-generation pharmaceutical heparin.

Association of ß-catenin with P-Smad3 but not LEF-1 dissociates in vitro profibrotic from anti-inflammatory effects of TGF-ß1.

Transforming growth factor ß1 (TGF-ß1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-ß/Smad and Wnt/ß-catenin pathways in epithelial-mesenchymal transition (EMT) suggests a specific role for ß-catenin in profibrotic effects of TGF-ß1. However, no such mechanistic role has been demonstrated for ß-catenin in the anti-inflammatory effects of TGF-ß1. In the present study, we explored the role of ß-catenin in the profibrotic and anti-inflammatory effects of TGF-ß1 by using a cytosolic, but not membrane, ß-catenin knockdown chimera (F-TrCP-Ecad) and the ß-catenin/CBP inhibitor ICG-001. TGF-ß1 induced nuclear Smad3/ß-catenin complex, but not ß-catenin/LEF-1 complex or TOP-flash activity, during EMT of C1.1 (renal tubular epithelial) cells. F-TrCP-Ecad selectively degraded TGF-ß1-induced cytoplasmic ß-catenin and blocked EMT of C1.1 cells. Both F-TrCP-Ecad and ICG-001 blocked TGF-ß1-induced Smad3/ß-catenin and Smad reporter activity in C1.1 cells, suggesting that TGF-ß1-induced EMT depends on ß-catenin binding to Smad3, but not LEF-1 downstream of Smad3, through canonical Wnt. In contrast, in J774 macrophages, the ß-catenin level was low and was not changed by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) with or without TGF-ß1. TGF-ß1 inhibition of LPS-induced TNF-α and IFN-γ-stimulated inducible NO synthase (iNOS) expression was not affected by F-TrCP-Ecad, ICG-001 or by overexpression of wild-type ß-catenin in J774 cells. Inhibition of ß-catenin by either F-TrCP-Ecad or ICG-001 abolished LiCl-induced TOP-flash, but not TGF-ß1-induced Smad reporter, activity in J774 cells. These results demonstrate for the first time that ß-catenin is required as a co-factor of Smad in TGF-ß1-induced EMT of C1.1 epithelial cells, but not in TGF-ß1 inhibition of macrophage activation. Targeting ß-catenin may dissociate the TGF-ß1 profibrotic and anti-inflammatory effects.

Mast cells produce novel shorter forms of perlecan that contain functional endorepellin: a role in angiogenesis and wound healing.

Mast cells are derived from hematopoietic progenitors that are known to migrate to and reside within connective and mucosal tissues, where they differentiate and respond to various stimuli by releasing pro-inflammatory mediators, including histamine, growth factors, and proteases. This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL-2H3 and HMC-1, produce the heparan sulfate proteoglycan, perlecan, with a molecular mass of 640 kDa as well as smaller molecular mass species of 300 and 130 kDa. Utilizing domain-specific antibodies coupled with N-terminal sequencing, it was confirmed that both forms contained the C-terminal module of the protein core known as endorepellin, which were generated by mast cell-derived proteases. Domain-specific RT-PCR experiments demonstrated that transcripts corresponding to domains I and V, including endorepellin, were present; however, mRNA transcripts corresponding to regions of domain III were not present, suggesting that these cells were capable of producing spliced forms of the protein core. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the α(2)ß(1) integrin and stimulate the migration of cells in "scratch assays," both activities of which were inhibited by incubation with either anti-endorepellin or anti-perlecan antibodies. This study shows for the first time that mast cells secrete and process the extracellular proteoglycan perlecan into fragments containing the endorepellin C-terminal region that regulate angiogenesis and matrix turnover, which are both key events in wound healing.

Pre-clinical lung squamous cell carcinoma mouse models to identify novel biomarkers and therapeutic interventions.

Primary lung carcinoma or lung cancer (LC) is classified into small-cell or non-small-cell (NSCLC) lung carcinoma. Lung squamous cell carcinoma (LSCC) is the second most common subtype of NSCLC responsible for 30% of all LCs, and its survival remains low with only 24% of patients living for five years or longer post-diagnosis primarily due to the advanced stage of tumors at the time of diagnosis. The pathogenesis of LSCC is still poorly understood and has hampered the development of effective diagnostics and therapies. This review highlights the known risk factors, genetic and epigenetic alterations, miRNA biomarkers linked to the development and diagnosis of LSCC and the lack of therapeutic strategies to target specifically LSCC. We will also discuss existing animal models of LSCC including carcinogen induced, transgenic and xenograft mouse models, and their advantages and limitations along with the chemopreventive studies and molecular studies conducted using them. The importance of developing new and improved mouse models will also be discussed that will provide further insights into the initiation and progression of LSCC, and enable the identification of new biomarkers and therapeutic targets.

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices.

BACKGROUND: The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. RESULTS: We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. CONCLUSION: Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

The absence of Brm exacerbates photocarcinogenesis.

Brm is an ATPase subunit of the SWI/SNF chromatin-remodelling complex. Previously, we identified a novel hotspot mutation in Brm in human skin cancer, which is caused by exposure to ultraviolet radiation (UVR). As SWI/SNF is involved in DNA repair, we investigated whether Brm-/- mice had enhanced photocarcinogenesis. P53+/- and Brm-/-p53+/- mice were also examined as the p53 tumor suppressor gene is mutated early during human skin carcinogenesis. Mice were exposed to a low-dose irradiation protocol that caused few skin tumors in wild-type mice. Brm-/- mice with both p53 alleles intact had an increased incidence of skin and ocular tumors compared to Brm+/+p53+/+ controls. Brm loss in p53+/- mice did not further enhance skin or ocular cancer incidence beyond the increased photocarcinogenesis in p53+/- mice. However, the skin tumors that arose early in Brm-/- p53+/- mice had a higher growth rate. Brm-/- did not prevent UVR-induced apoptotic sunburn cell formation, which is a protective response. Unexpectedly, Brm-/- inhibited UVR-induced immunosuppression, which would be predicted to reduce rather than enhance photocarcinogenesis. In conclusion, the absence of Brm increased skin and ocular photocarcinogenesis. Even when one allele of p53 is lost, Brm has additional tumor suppressing capability.

Intravital imaging technology guides FAK-mediated priming in pancreatic cancer precision medicine according to Merlin status.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow­induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

Regional Variation in Epidermal Susceptibility to UV-Induced Carcinogenesis Reflects Proliferative Activity of Epidermal Progenitors.

To better understand the influence of ultraviolet (UV) irradiation on the initial steps of skin carcinogenesis, we examine patches of labeled keratinocytes as a proxy for clones in the interfollicular epidermis (IFE) and measure their size variation upon UVB irradiation. Multicolor lineage tracing reveals that in chronically irradiated skin, patches near hair follicles (HFs) increase in size, whereas those far from follicles do not change. This is explained by proliferation of basal epidermal cells within 60 µm of HF openings. Upon interruption of UVB, patch size near HFs regresses significantly. These anatomical differences in proliferative behavior have significant consequences for the cell of origin of basal cell carcinomas (BCCs). Indeed, a UV-inducible murine BCC model shows that BCC patches are more frequent, larger, and more invasive near HFs. These findings have major implications for the prevention of field cancerization in the epidermis.

Mesangial cell-derived factors alter monocyte activation and function through inflammatory pathways: possible pathogenic role in diabetic nephropathy.

Infiltration of macrophages to the kidney is a feature of early diabetic nephropathy. For this to happen monocytes must become activated, migrate from the circulation, and infiltrate the mesangium. This process involves degradation of extracellular matrix, a process mediated by matrix metalloproteinases (MMPs). In the present study we investigate the expression of proinflammatory cytokines TNF-alpha, IL-6, and MMP-9 in glomeruli of control and diabetic rodents and use an in vitro coculture system to examine whether factors secreted by mesangial cells in response to a diabetic milieu can induce monocyte MMP-9 expression and infiltration. After 8 wk of diabetes, the glomerular level of TNF-alpha, IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P < 0.01). Coculture of THP1 monocytes and glomerular mesangial cells in 5 or 25 mM glucose increased MMP-9 (5 mM: 65% and 25 mM: 112%; P < 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P < 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP1 cells. High glucose (25 mM) increased TNF-alpha, IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P < 0.05), but only TNF-alpha and IL-6 increased MMP-9 expression (50- and 60-fold, respectively; P < 0.05). Our results show that mesangial cell-secreted factors increase monocyte adhesion, differentiation, MMP expression, and degradative capacity. High glucose could augment these effects by increasing mesangial cell proinflammatory cytokine secretion. This mesangial cell-monocyte interaction may be important in activating monocytes to migrate from the circulation to the kidney in the early stages of diabetic nephropathy.

Visualizing the Contribution of Keratin-14+ Limbal Epithelial Precursors in Corneal Wound Healing.

It is thought that corneal epithelial injuries resolve by leading-edge cells "sliding" or "rolling" into the wound bed. Here, we challenge this notion and show by real-time imaging that corneal wounds initially heal by "basal cell migration." The K14CreERT2-Confetti multi-colored reporter mouse was employed to spatially and temporally fate-map cellular behavior during corneal wound healing. Keratin-14+ basal epithelia are forced into the wound bed by increased population pressure gradient from the limbus to the wound edge. As the defect resolves, centripetally migrating epithelia decelerate and replication in the periphery is reduced. With time, keratin-14+-derived clones diminish in number concomitant with their expansion, indicative that clonal evolution aligns with neutral drifting. These findings have important implications for the involvement of stem cells in acute tissue regeneration, in key sensory tissues such as the cornea.

Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC.

Clonal diversity in carcinomas: its implications for tumour progression and the contribution made to it by epithelial-mesenchymal transitions.

The progression of tumours to malignancy is commonly considered to arise through lineal evolution, a process in which mutations conferring pro-oncogenic cellular phenotypes are acquired by a succession of ever-more dominant clones. However, this model is at odds with the persistent polyclonality observed in many cancers. We propose that an alternative mechanism for tumour progression, called interclonal cooperativity, is likely to play a role at stages of tumour progression when mutations cause microenvironmental changes, such as occur with epithelial-mesenchymal transitions (EMTs). Interclonal cooperativity occurs when cancer cell-cancer cell interactions produce an emergent malignant phenotype from individually non-malignant clones. In interclonal cooperativity, the oncogenic mutations occur in different clones within the tumour that complement each other and cooperate in order to drive progression. This reconciles the accepted genetic and evolutionary basis of cancers with the observed polyclonality in tumours. Here, we provide a conceptual basis for examining the importance of cancer cell-cancer cell interactions to the behaviour of tumours and propose specific mechanisms by which clonal diversity in tumours, including that provided by EMTs, can drive the progression of tumours to malignancy.

Inflammatory doses of UV may not be necessary for skin carcinogenesis.

The UV wavelengths in sunlight are the main cause of skin cancer in humans. Sunlight causes gene mutations, immunosuppression and, at higher doses, inflammation. While it is clear that immunosuppression and gene mutations are essential biologic events via which UV causes skin cancer, the requirement for UV-induced inflammation is less certain. Both the UVB (290-320 nm) and UVA (320-400 nm) wavebands within sunlight can cause skin cancer, gene mutations and immunosuppression. However, UVB, but not UVA, at realistic doses can cause inflammation, and UVB induces skin cancer, immunosuppression and gene mutations at doses much lower than those required to cause inflammation. Inflammation enhances skin carcinogenesis, but may not be UV induced, and inflammatory mediators at doses too low to cause inflammation may be required. UV-induced mutations can cause epidermal cells to make proinflammatory factors or to induce them in the surrounding stroma, creating an oxidizing environment in which additional oncogenic mutations are likely to take place, even in the absence of UV. Our hypothesis is therefore that subinflammatory doses of both UVA and UVB cause benign skin tumors. One of the effects of sunlight-induced mutations may be the production of inflammatory mediators that enhance carcinogenesis.

Brahma deficiency in keratinocytes promotes UV carcinogenesis by accelerating the escape from cell cycle arrest and the formation of DNA photolesions.

BACKGROUND: Ultraviolet radiation (UVR) is the principal cause of keratinocyte skin cancers. Previous work found that levels of the chromatin remodelling protein, Brahma (Brm), are diminished during the progression from actinic keratoses to cutaneous squamous cell carcinomas in humans, and its loss in UV-irradiated mouse skin causes epidermal hyperplasia and increased tumour incidence. METHODS: The skins of mice and mouse and human keratinocytes deficient in Brm were exposed to UVR and evaluated for cell cycle progression and DNA damage response. OBJECTIVE: To identify the mechanisms by which loss of Brm contributes to UVR-induced skin carcinogenesis. RESULTS: In both mouse keratinocytes and HaCaT cells, Brm deficiency led to an increased cell population growth following UVR exposure compared to cells with normal levels of Brm. Cell cycle analysis using a novel assay showed that Brm-deficient keratinocytes entered cell cycle arrest normally, but escaped from cell cycle arrest faster, enabling them to begin proliferating earlier. In mouse keratinocytes, Brm primarily affected accumulation in G0/G1-phase, whereas in HaCaT cells, which lack normal p53, accumulation in G2/M-phase was affected. Brm-deficient keratinocytes in mouse skin and human cell cultures also had higher levels of UVR-induced cyclobutane pyrimidine dimer photolesions. These effects occurred without any compensatory increase in DNA repair or cell death to remove photolesions or the cells that harbor them from the keratinocyte population. CONCLUSION: The loss of Brm in keratinocytes exposed to UVR enables them to resume proliferation while harboring DNA photolesions, leading to an increased fixation of mutations and, consequently, increased carcinogenesis.

Sensitization of prostate cancer to radiation therapy: Molecules and pathways to target.

Radiation therapy is used to treat cancer by radiation-induced DNA damage. Despite the best efforts to eliminate cancer, some cancer cells survive irradiation, resulting in cancer progression or recurrence. Alteration in DNA damage repair pathways is common in cancers, resulting in modulation of their response to radiation. This article focuses on the recent findings about molecules and pathways that potentially can be targeted to sensitize prostate cancer cells to ionizing radiation, thereby achieving an improved therapeutic outcome.

Human papillomavirus 16 E6 modulates the expression of miR-496 in oropharyngeal cancer.

Human papillomavirus (HPV), notably type 16, is a risk factor for up to 75% of oropharyngeal squamous cell carcinomas (SCC). It has been demonstrated that small non-coding RNAs known as microRNAs play a vital role in the cellular transformation process. In this study, we used an LNA array to further investigate the impact of HPV16 on the expression of microRNAs in oropharyngeal (tonsillar) cancer. A number of miRNAs were found to be deregulated, with miR-496 showing a four-fold decrease. Over-expression of the high risk E6 oncoprotein down-regulated miR-496, impacting upon the post-transcriptional control of the transcription factor E2F2. These HPV specific miRNAs were integrated with the HPV16 interactome to identify possible mechanistic pathways. These analyses provide insights into novel molecular interactions between HPV16 and miRNAs in oropharyngeal cancers.