MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

Abstract Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.

Leptin receptor 170 kDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance

[EN] To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2-dependent leptin signalling and signal transducer and activator of transcription 3 (STAT3) phosphorylation and reduce AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Deltoid and vastus lateralis muscle biopsies were obtained from 20 men: 10 non-obese control subjects (mean +/- s.d. age, 31 +/- 5 years; height, 184 +/- 9 cm; weight, 91 +/- 13 kg; and percentage body fat, 24.8 +/- 5.8%) and 10 obese (age, 30 +/- 7 years; height, 184 +/- 8 cm; weight, 115 +/- 8 kg; and percentage body fat, 34.9 +/- 5.1%). Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P < 0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPKalpha and ACCbeta phosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPKalpha and ACCbeta phosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P < 0.05). In summary, skeletal muscle leptin receptors and leptin signalling are reduced in obesity, particularly in the leg muscles.

Human papillomavirus (HPV) and associated diseases: between applied diagnostic and basic research

Human Papillomavirus (HPV) is the cause of cervical cancers (among these, adenocarcinoma, AdCa) and is associated to a subgroup of oropharyngeal carcinomas (OPSCCs). Even if the risk for cancer development is linked to the infection by some viral genotypes, mainly HPV16 and 18, viral DNA alone seems not to be sufficient for diagnosis. Moreover, the role of the virus in OPSCCs has not been totally clarified yet. In the first part of the thesis, the performances concerning viral genotyping in clinical cervical samples of a new pyrosequencing-based test and a well-known hybridization-based assay have been compared. Similar results between the methods have been obtained. However, the former showed advantages in detecting intratype variants, higher specificity and a broader spectrum of detectable HPV types. The second part deals with the evaluation of virological markers (genotyping, viral oncoproteins expression, viral load, physical state and CpG methylation of HPV16 genome) in the diagnosis/prognosis of cervical AdCa and HPV-associated OPSCCs. HPV16 has been confirmed the most prevalent genotype in both the populations. Interestingly, the mean methylation frequency of viral DNA at the early promoter showed the tendency to be associated to invasion for cervical AdCa and to a worse prognosis for OPSCCs, suggesting a promising role as diagnostic/prognostic biomarker. The experiments of the third part were performed at the DKFZ in Heidelberg (Germany) and dealt with the analysis of the response to IFN-k transfection in HPV16-positive cervical cancer and head&neck carcinoma cell lines to evaluate its potential role as new treatment. After 24h, we observed increased IFN-b expression which lead to the up-regulation of genes involved in the antigens presentation pathway (MHC class I and immunoproteasome) and antiviral response as well, in particular in cervical cancer cell lines. This fact suggested also the presence of different HPV-mediated carcinogenic pathways between the two anatomical districts.

Musculoskeletal tissue regeneration by human non-embryonic stem cells

The aim of this thesis was to investigate the regenerative potential of alternative sources of stem cells, derived from human dental pulp (hDPSCs) and amniotic fluid (hAFSCs) and, specifically, to evaluate their capability to be committed towards osteogenic and myogenic lineages, for the eventual applicability of these stem cells to translational strategies in regenerative medicine of bone and skeletal muscle tissues. The in vitro bone production by stem cells may represent a radical breakthrough in the treatment of pathologies and traumas characterized by critical bone mass defects, with no medical or surgical solution. Human DPSCs and AFSCs were seeded and pre-differentiated on different scaffolds to test their capability to subsequently reach the osteogenic differentiation in vivo, in order to recover critical size bone defects. Fibroin scaffold resulted to be the best scaffold promoting mature bone formation and defect correction when combined to both hDPSCs and hAFSCs. This study also described a culture condition that might allow human DPSCs to be used for human cell therapy in compliance with good manufacturing practices (GMPs): the use of human serum (HS) promoted the expansion and the osteogenic differentiation of hDPSCs in vitro and, furthermore, allowed pre-differentiated hDPSCs to regenerate critical size bone defects in vivo. This thesis also showed that hDPSCs and hAFSCs can be differentiated towards the myogenic lineage in vitro, either when co-cultured with murine myoblasts and when differentiated alone after DNA demethylation treatment. Interestingly, when injected into dystrophic muscles of SCID/mdx mice - animal model of Duchenne Muscular Dystrophy (DMD) - hDPSCs and hAFSCs pre-differentiated after demethylating treatment were able to regenerate the skeletal muscle tissue and, particularly, to restore dystrophin expression. These observations suggest that human DPSCs and AFSCs might be eventually applied to translational strategies, in order to enhance the repair of injured skeletal muscles in DMD patients.

Diversity of the proximal femur in humans: morphological variations of the head-neck junction

The proximal femur is a high-diversity region of the human skeleton, especially at the anterior junction between head and neck, where various bony morphologies have been recognized since mid nineteenth century. Classical literature on this topic is chaotic and contradictory, making almost impossible the comparison of data from different researches. Starting from an extensive bibliographic review, the first standardized method to score these traits has been created. This method allows representing both the anatomical diversity of the region already described in literature and a part of variability not considered before, giving few and univocal definitions and allowing to collect comparable data. The method has been applied to three identified and five archaeological European skeletal collections, with the aim of investigating the distribution of these features by sex, age and side, in different places and time periods. It has also been applied to 3D digital reconstructions of femurs from CT scan files of coxo-femoral joints from fresh cadavers. In addition to the osseous traits described in the standardized method, the presence and frequency of some features known as herniation pits have been scored both on bones and on CT scans. The various osseous traits of the proximal femur are present at similar frequencies in skeletal samples from different countries and different historical periods, even if with clear local differentiation. Some of the features examined show significant trends related to their distribution by gender and age. Some hypotheses are proposed about the etiology of these morphologies and their possible implication with the acquisition of bipedalism in Humans. It is therefore highlighted the possible relation of some of these traits with the development of disorders of the hip joint. Moreover, it is not recommended the use of any of these features as a specific activity-related marker.

[Bullet and shrapnel injuries in the face and neck regions. Current aspects of wound ballistics]

A basic understanding of the ballistic behaviour of projectiles or fragments after entering the human body is essential for the head and neck surgeon in the military environment in order to anticipate the diagnostic and therapeutic consequences of this type of injury. Although a large number of factors influence the missile in flight and after penetration of the body, the most important factor is the amount of energy transmitted to the tissue. Long guns (rifles or shotguns) have a much higher muzzle energy compared to handguns, explaining why the remote effects beyond the bullet track play a major role. While most full metal jacket bullets release their energy after 12-20 cm (depending on the calibre), soft point bullets release their energy immediately after entry into the human body. This results in a major difference in extremity wounds, but not so much in injuries with long bullet paths (e.g. diagonal shots). Shrapnel wounds are usually produced with similarly high kinetic energy to those caused by hand- and long guns. However, fragments tend to dissipate the entire amount of energy within the body, which increases the degree of tissue disruption. Of all relevant injuries in the head and neck region, soft tissue injuries make up the largest proportion (60%), while injuries to the face are seen three times more often than injuries to the neck. Concomitant intracranial or spinal injury is seen in 30% of cases. Due to high levels of wound contamination, the infection rate is approximately 15%, often associated with a complicated and/or multiresistant spectrum of germs.

Magnetization exchange observed in human skeletal muscle by non-water-suppressed proton magnetic resonance spectroscopy

Many metabolites in the proton magnetic resonance spectrum undergo magnetization exchange with water, such as those in the downfield region (6.0-8.5 ppm) and the upfield peaks of creatine, which can be measured to reveal additional information about the molecular environment. In addition, these resonances are attenuated by conventional water suppression techniques complicating detection and quantification. To characterize these metabolites in human skeletal muscle in vivo at 3 T, metabolite cycled non-water-suppressed spectroscopy was used to conduct a water inversion transfer experiment in both the soleus and tibialis anterior muscles. Resulting median exchange-independent T(1) times for the creatine methylene resonances were 1.26 and 1.15 s, and for the methyl resonances were 1.57 and 1.74 s, for soleus and tibialis anterior muscles, respectively. Magnetization transfer rates from water to the creatine methylene resonances were 0.56 and 0.28 s(-1) , and for the methyl resonances were 0.39 and 0.30 s(-1) , with the soleus exhibiting faster transfer rates for both resonances, allowing speculation about possible influences of either muscle fibre orientation or muscle composition on the magnetization transfer process. These water magnetization transfer rates observed without water suppression are in good agreement with earlier reports that used either postexcitation water suppression in rats, or short CHESS sequences in human brain and skeletal muscle.

Patients with temporomandibular disorders have increased fatigability of the cervical extensor muscles

To determine whether patients with myogenous and mixed temporomandibular disorders (TMD) have greater fatigability of the cervical extensor muscles while performing a neck extensor muscle endurance test (NEMET) when compared with healthy controls.

Repetitive TMS over the human oculomotor cortex: comparison of 1-Hz and theta burst stimulation

The aim of the study was to compare the effect duration of two different protocols of repetitive transcranial magnetic stimulation (rTMS) on saccade triggering. In four experiments, two regions (right frontal eye field (FEF) and vertex) were stimulated using a 1-Hz and a theta burst protocol (three 30Hz pulses repeated at intervals of 100ms). The same number of TMS pulses (600 pulses) was applied with stimulation strength of 80% of the resting motor threshold for hand muscles. Following stimulation the subjects repeatedly performed an oculomotor task using a modified overlap paradigm, and saccade latencies were measured over a period of 60min. The results show that both 1-Hz and theta burst stimulation had inhibitory effects on saccade triggering when applied over the FEF, but not over the vertex. One-hertz rTMS significantly increased saccade latencies over a period of about 8min. After theta burst rTMS, this effect lasted up to 30min. Furthermore, the decay of rTMS effects was protocol-specific: After 1-Hz stimulation, saccade latencies returned to a baseline level much faster than after theta burst stimulation. We speculate that these time course differences represent distinct physiological mechanisms of how TMS interacts with brain function.

Human experimental pain models in drug development: translational pain research

Human experimental pain models require standardized stimulation and quantitative assessment of the evoked responses. This approach can be applied to healthy volunteers and pain patients before and after pharmacological interventions. Standardized stimuli of different modalities (ie, mechanical, chemical, thermal or electrical) can be applied to the skin, muscles and viscera for a differentiated and comprehensive assessment of various pain pathways and mechanisms. Using a multi-modal, multi-tissue approach, new and existing analgesic drugs can be profiled by their modulation of specific biomarkers. It has been shown that biomarkers, for example, those related to the central integration of repetitive nociceptive stimuli, can predict efficacy of a given drug in neuropathic pain conditions. Human experimental pain models can bridge animal and clinical pain research, and act as translational research providing new possibilities for designing successful clinical trials. Proof-of-concept studies provide cheap, fast and reliable information on dose-efficacy relationships and how pain sensed in the skin, muscles and viscera are inhibited.

Mapping of direction and muscle representation in the human primary motor cortex controlling thumb movements

Larger body parts are somatotopically represented in the primary motor cortex (M1), while smaller body parts, such as the fingers, have partially overlapping representations. The principles that govern the overlapping organization of M1 remain unclear. We used transcranial magnetic stimulation (TMS) to examine the cortical encoding of thumb movements in M1 of healthy humans. We performed M1 mapping of the probability of inducing a thumb movement in a particular direction and used low intensity TMS to disturb a voluntary thumb movement in the same direction during a reaction time task. With both techniques we found spatially segregated representations of the direction of TMS-induced thumb movements, thumb flexion and extension being best separated. Furthermore, the cortical regions corresponding to activation of a thumb muscle differ, depending on whether the muscle functions as agonist or as antagonist for flexion or extension. In addition, we found in the reaction time experiment that the direction of a movement is processed in M1 before the muscles participating in it are activated. It thus appears that one of the organizing principles for the human corticospinal motor system is based on a spatially segregated representation of movement directions and that the representation of individual somatic structures, such as the hand muscles, overlap.

DXA predictions of human femoral mechanical properties depend on the load configuration

The aim of this study was to evaluate the ability of dual energy X-rays absorptiometry (DXA) areal bone mineral density (aBMD) measured in different regions of the proximal part of the human femur for predicting the mechanical properties of matched proximal femora tested in two different loading configurations. 36 pairs of fresh frozen femora were DXA scanned and tested until failure in two loading configurations: a fall on the side or a one-legged standing. The ability of the DXA output from four different regions of the proximal femur in predicting the femoral mechanical properties was measured and compared for the two loading scenarios. The femoral neck DXA BMD was best correlated to the femoral ultimate force for both configurations and predicted significantly better femoral failure load (R2=0.80 vs. R2=0.66, P<0.05) when simulating a side than when simulating a standing configuration. Conversely, the work to failure was predicted similarly for both loading configurations (R2=0.54 vs. R2=0.53, P>0.05). Therefore, neck BMD should be considered as one of the key factors for discriminating femoral fracture risk in vivo. Moreover, the better predictive ability of neck BMD for femoral strength if tested in a fall compared to a one-legged stance configuration suggests that DXA's clinical relevance may not be as high for spontaneous femoral fractures than for fractures associated to a fall.

Plakins, a Versatile Family of Cytolinkers: Roles in Skin Integrity and in Human Diseases

The plakin family consists of giant proteins involved in the cross-linking and organization of the cytoskeleton and adhesion complexes. They further modulate several fundamental biological processes, such as cell adhesion, migration, and polarization or signaling pathways. Inherited and acquired defects of plakins in humans and in animal models potentially lead to dramatic manifestations in the skin, striated muscles, and/or nervous system. These observations unequivocally demonstrate the key role of plakins in the maintenance of tissue integrity. Here we review the characteristics of the mammalian plakin members BPAG1 (bullous pemphigoid antigen 1), desmoplakin, plectin, envoplakin, epiplakin, MACF1 (microtubule-actin cross-linking factor 1), and periplakin, highlighting their role in skin homeostasis and diseases.

Characterization and subchromosomal location of a novel tumor suppressor gene on human chromosome 7

Alterations in oncogenes and tumor suppressor genes (TSGs) are considered to be critical steps in oncogenesis. Consistent deletions and loss of heterozygosity (LOH) of polymorphic markers in a determinate chromosomal fragment are known to be indicative of a closely mapping TSG. Deletion of the long arm of chromosome 7 (hchr 7) is a frequent trait in many kinds of human primary tumors. LOH was studied with an extensive set of markers on chromosome 7q in several types of human neoplasias (primary breast, prostate, colon, ovarian and head and neck carcinomas) to determine the location of a putative TSG. The extent of LOH varied depending the type of tumor studied but all the LOH curves we obtained had a peak at (C-A)$\sb{\rm n}$ microsatellite repeat D7S522 at 7q31.1 and showed a Gaussian distribution. The high incidence of LOH in all tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on the 7q31.1. To investigate whether the putative TSG is conserved in the syntenic mouse locus, we studied LOH of 30 markers along mouse chromosome 6 (mchr 6) in chemically induced squamous cell carcinomas (SCCs). Tumors were obtained from SENCAR and C57BL/6 x DBA/2 F1 females by a two-stage carcinogenesis protocol. The high incidence of LOH in the tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on mchr 6 A1. Since this segment is syntenic with the hchr 7q31, these data indicate that the putative TSG is conserved in both species. Functional evidence for the existence of a TSG in hchr 7 was obtained by microcell fusion transfer of a single hchr 7 into a murine SCC-derived cell line. Five out of seven hybrids had two to three-fold longer latency periods for in vivo tumorigenicity assays than parental cells. One of the unrepressed hybrids had a deletion in the introduced chromosome 7 involving q31.1-q31.3, confirming the LOH data. ^

Radiolabeled human monoclonal IgM for intracompartmental cancer therapy

Radioimmunotherapy (RIT) with i.v. administered radiolabeled IgG can selectively irradiate tumor cells in vivo. However, it only provides effective therapy for lymphomas. Intracompartmental RIT with radiolabeled human monoclonal IgM may allow curative treatment of solid tumors by increasing tumor deposition of radioactivity, reducing systemic toxicity and allowing repeated administration. This hypothesis was tested in nude mouse models with IgM radiolabeled with indium-111 $\rm(\sp{111}In)$ or yttrium-90 $\rm(\sp{90}Y).$ The use of two radioisotopes, $\rm\sp{111}In$ for imaging and $\rm\sp{90}Y$ for therapy, allow for more quantitative and cautious development of RIT.^ Radiolabled 2B12, an IgM reactive with human ovarian carcinomas was tested by i.v. and intraperitoneal (i.p.) administration in nude mice bearing i.p. nodules of a human ovarian carcinoma cell line (SKOV3 NMP2). Radiolabeled CR4E8, an IgM reactive with human squamous cell carcinomas was tested by i.v. and intralesional (i.l.) administration in nude mice bearing subcutaneous tumors of a human head and neck squamous cell carcinoma cell line (886). These two models were selected to test proof of concept. Radiolabeled irrelevant IgM (CH-1B9), and $\rm\sp{90}Y$-aggregate served as specificity controls. Biodistribution was performed by excising, weighing and then measuring the radioactivity of tumor and normal organs. Therapy was conducted with i.p. $\rm\sp{90}Y$-labeled 2B12 using both single and fractionated administration and with i.l. $\rm\sp{90}Y$-labeled CR4E8 using single administration. Mice were monitored for tumor response, survival and systemic toxicity.^ Intracompartmental administration of radiolabeled IgM produced immediate high and prolonged tumor deposition of radioactivity with low normal tissue uptake. In contrast, i.v. administration resulted in low tumor, but high liver and spleen uptake. Similar biodistributions were demonstrated for $\rm\sp{111}In$- and $\rm\sp{90}Y$-labeled IgM. Intraperitoneal therapy with $\rm\sp{90}Y$-labeled 2B12 increased survival by approximately 12 days for every 100 $\rm\mu Ci$ of activity without significant toxicity for single (0-300 $\rm\mu Ci)$ and fractionated (150-510 $\rm\mu Ci)$ administration. Intralesional therapy with $\rm\sp{90}Y$-labeled CR4E8 (150-400 $\rm\mu Ci)$ induced prolonged complete regressions. Significant local or systemic toxicity was not observed.^ Intracompartmental RIT with radiolabeled tumor-reactive human monoclonal IgM can selectively irradiate tumor cells. Intracompartmental radiolabled IgM can significantly extend the survival of treated mice with minimal toxicity. It deserves further development as a new cancer therapy. ^