Salivary microbiome changes distinguish response to chemoradiotherapy in patients with oral cancer.

BACKGROUND: Oral squamous cell carcinoma (SCC) is associated with oral microbial dysbiosis. In this unique study, we compared pre- to post-treatment salivary microbiome in patients with SCC by 16S rRNA gene sequencing and examined how microbiome changes correlated with the expression of an anti-microbial protein. RESULTS: Treatment of SCC was associated with a reduction in overall bacterial richness and diversity. There were significant changes in the microbial community structure, including a decrease in the abundance of Porphyromonaceae and Prevotellaceae and an increase in Lactobacillaceae. There were also significant changes in the microbial community structure before and after treatment with chemoradiotherapy, but not with surgery alone. In patients treated with chemoradiotherapy alone, several bacterial populations were differentially abundant between responders and non-responders before and after therapy. Microbiome changes were associated with a change in the expression of DMBT1, an anti-microbial protein in human saliva. Additionally, we found that salivary DMBT1, which increases after treatment, could serve as a post-treatment salivary biomarker that links to microbial changes. Specifically, post-treatment increases in human salivary DMBT1 correlated with increased abundance of Gemella spp., Pasteurellaceae spp., Lactobacillus spp., and Oribacterium spp. This is the first longitudinal study to investigate treatment-associated changes (chemoradiotherapy and surgery) in the oral microbiome in patients with SCC along with changes in expression of an anti-microbial protein in saliva. CONCLUSIONS: The composition of the oral microbiota may predict treatment responses; salivary DMBT1 may have a role in modulating the oral microbiome in patients with SCC. After completion of treatment, 6 months after diagnosis, patients had a less diverse and less rich oral microbiome. Leptotrichia was a highly prevalent bacteria genus associated with disease. Expression of DMBT1 was higher after treatment and associated with microbiome changes, the most prominent genus being Gemella Video Abstract.

Unearthing the inhibitory potential of phytochemicals from Lawsonia inermis L. and some drugs against dengue virus protein NS1: an in silico approach.

Dengue has received the status of an epidemic and endemic disease, with countless number of infections every year. Due to the unreliability of vaccines and non-specificity of drugs, it becomes necessary to find plant-based alternatives, with less harmful side effects. Lawsonia inermis L., is the sole source of dye, Mehendi. The rich repertoire of phytochemicals makes it useful, medicinally. The main objectives of the study are to explore the anti-dengue properties of the phytochemicals from Lawsonia inermis, and to shortlist potential candidates in curing the disease. Phytochemicals from the plant, and a set of drugs were screened and docked against NS1 protein, a less explored drug target, needed for maintenance of virus life cycle. Ligand screening and docking analysis concluded gallic acid, and chlorogenic acid to be good candidates, exhibiting high binding affinity and extensive interactions with the protein. From among the shortlisted drugs, only Vibegron showed effective binding affinity with NS1 protein with zero violations to the Lipinski's Rule of 5. Molecular dynamic simulations, executed for a time period of 100 nanoseconds, reveal the performance of a ligand within a solvated system. Chlorogenic and gallic acid, formed more stable and compact complexes with protein, with stable energy parameters and strong binding affinity. This was further validated with snapshots taken every 50 nanoseconds, showing no change in binding site between the ligand and protein, within the stipulated time frame. It was interesting to see that, a phenol (chlorogenic acid), served as a better drug candidate, against the NS1 protein.Communicated by Ramaswamy H. Sarma.

Elucidating the structure-function attributes of a trypanosomal arginyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.

Sudden Unexpected Death in Epilepsy and Respiratory Defects in a Mouse Model of DEPDC5-Related Epilepsy.

OBJECTIVES: DEPDC5 is a common causative gene in familial focal epilepsy with or without malformations of cortical development. Its pathogenic variants also confer a significantly higher risk for sudden unexpected death in epilepsy (SUDEP), providing opportunities to investigate the pathophysiology intersecting neurodevelopment, epilepsy, and cardiorespiratory function. There is an urgent need to gain a mechanistic understanding of DEPDC5-related epilepsy and SUDEP, identify biomarkers for patients at high risk, and develop preventive interventions. METHODS: Depdc5 was specifically deleted in excitatory or inhibitory neurons in the mouse brain to determine neuronal subtypes that drive epileptogenesis and SUDEP. Electroencephalogram (EEG), cardiac, and respiratory recordings were performed to determine cardiorespiratory phenotypes associated with SUDEP. Baseline respiratory function and the response to hypoxia challenge were also studied in these mice. RESULTS: Depdc5 deletion in excitatory neurons in cortical layer 5 and dentate gyrus caused frequent generalized tonic-clonic seizures and SUDEP in young adult mice, but Depdc5 deletion in cortical interneurons did not. EEG suppression immediately following ictal offset was observed in fatal and non-fatal seizures, but low amplitude rhythmic theta frequency activity was lost only in fatal seizures. In addition, these mice developed baseline respiratory dysfunction prior to SUDEP, during which ictal apnea occurred long before terminal cardiac asystole. INTERPRETATION: Depdc5 deletion in excitatory neurons is sufficient to cause DEPDC5-related epilepsy and SUDEP. Ictal apnea and respiratory dysregulation play critical roles in SUDEP. Our study also provides a novel mouse model to investigate the underlying mechanisms of DEPDC5-related epilepsy and SUDEP. ANN NEUROL 2023;94:812-824.

COPD and glycopyrronium responsiveness assessment: An appraisal.

Clinical Trial Registration: ECR/159/Inst/WB/2013/RR-20. Background: Glycopyrronium bromide (a long-acting antimuscarinic agent: LAMA) appears pharmacokinetically suitable for testing bronchodilator responsiveness as salbutamol (short-acting ß2-agonist: SABA). Exploring the feasibility, acceptability, degree of reversibility with glycopyrronium, and its comparison with that of salbutamol may be intriguing. Methods: New, consecutive, and willing outpatient attendees in the same season of the two consecutive years with chronic obstructive pulmonary disease (FEV1/FVC <0.07; FEV1 <80% of predicted) were subjected to serial responsiveness with inhalation of salbutamol first followed by 50 µg dry powder glycopyrronium [Salbutamol- Glycopyrronium] (phase-1) in the first year and glycopyrronium followed by salbutamol [Glycopyrronium- Salbutamol] (phase-2) in the following year. We looked for the acceptability, adverse reactions, and degree of changes in FEV1, FVC, FEV1/FVC, and FEF25-75 with comparison between the two groups. Results: The [Salbutamol- Glycopyrronium] group (n = 86) were similar in age, body mass index, and FEV1 to the [Glycopyrronium- Salbutamol] group (n = 88). Both the agents could make a significant (P <.0001) improvement in the parameters independently or as add-on when used serially in alternate orders. The intergroup difference at no stage was significant. The sensitive patients to salbutamol (n = 48), glycopyrronium (n = 44), and both (n = 12) have improvement of 165, 189, and 297 mL while a both-insensitive group (n = 70) had barely 44 mL of improvement. The protocol was universally accepted without any adverse events. Conclusion: Serial testing of salbutamol and glycopyrronium responsiveness in alternate orders provides an insight regarding the independent and the add-on effects of these two agents. About 40% of our chronic obstructive pulmonary disease patients had no clinically appreciable difference in FEV1 with the salbutamol + glycopyrronium combination inhalation.

Galanin mediates tumor-induced immunosuppression in head and neck squamous cell carcinoma.

PURPOSE: Galanin receptor 2 (GALR2) plays a significant role in the progression of head and neck squamous cell carcinomas (HNSCC). Since there is virtually no information on immunomodulation mediated by its ligand in the tumor microenvironment, we assessed the effects of galanin on peripheral blood mononuclear cells (PBMCs). METHODS: After verification of GALR2 expression and it activity in PBMCs we evaluated the effect of galanin and conditioned media from HNSCC cell lines silenced for galanin or antibody-depleted, on proliferation, apoptosis, cytokine expression and activation/differentiation of immune cells. RESULTS: We found that galanin alone and as a component of the HNSCC secretome decreased HNSCC cell proliferation and expression of pro-inflammatory cytokines (IFNγ, IL-12, IL-17A, IL-1α, IL-6 and TNF-α), whilst increasing apoptosis and expression of pro-tumoral cytokines/growth factors (IL-10, IL-4, PDGF and GM-CSF). T cell activation (using CD69 as activation marker) and anti-tumoral phenotypes in CD4+ T cells (Th1 and Th17) were found to be suppressed. In vivo, tumor growth was found to be increased in the presence of galanin-stimulated PBMCs. Data from The Cancer Genome Atlas (TCGA) revealed that high expression of galanin was associated with a reduced overall survival of patients with HNSCC. CONCLUSION: Our data indicate that galanin secreted by HNSCC cells exhibits immune-suppressive and pro-tumoral effects.

First report of gut bacterial dataset of a tribal Bhutia family from West Bengal, India.

The tribes of West Bengal are distributed in geographically distinct regions with distinctive features of their habitats and many of these tribes still practice a traditional livelihood avoiding the western diet. Hence, it is expected that their gut should remain pristine. In this study, we report the gut bacterial abundance of a Drukpa Bhutia tribal family of Lepchakha, inhabiting the hilly terrain of the Buxa region of Alipurduar district. First fecal matter was collected followed by Illumina Hiseq sequencing. Following standard protocols for metagenomic analysis, quality control (FASTQC), taxonomic profiling (QIIME, KRONA) and pathogenic load analysis were performed. This study revealed a set of core gut bacteria among which Bacteroides was identified to be the most abundant followed by Bifidobacterium, Streptococcus etc. Genera exhibiting lowest abundance were Eggerthella, Ruminococcus, Enterococcus etc. among the male, kid and female respectively. This data provides important insights into the distribution of bacterial members under study.

Aminoacyl-tRNA Synthetases, Indispensable Players in Lung Tumorigenesis.

Being an essential enzyme in protein synthesis, the aminoacyl-tRNA synthetases (aaRSs) have a conserved function throughout evolution. However, research has uncovered altered expressions as well as interactions of aaRSs, in league with aaRS-interacting multi-functional proteins (AIMPs), forming a multi-tRNA synthetase complex (MSC) and divulging into their roles outside the range of protein synthesis. In this review, we have directed our focus into the rudimentary structure of this compact association and also how these aaRSs and AIMPs are involved in the maintenance and progression of lung cancer, the principal cause of most cancer-related deaths. There is substantial validation that suggests the crucial role of these prime housekeeping proteins in lung cancer regulation. Here, we have addressed the biological role that the three AIMPs and the aaRSs play in tumorigenesis, along with an outline of the different molecular mechanisms involved in the same. In conclusion, we have introduced the potentiality of these components as possible therapeutics for the evolution of new-age treatments of lung tumorigenesis.

Phosphorylation of TRIP13 at Y56 induces radiation resistance but sensitizes head and neck cancer to cetuximab.

Radiation therapy, a mainstay of treatment for head and neck cancer, is not always curative due to the development of treatment resistance; additionally, multi-institutional trials have questioned the efficacy of concurrent radiation with cetuximab, the epidermal growth factor receptor (EGFR) inhibitor. We unraveled a mechanism for radiation resistance; that is, radiation induces EGFR, which phosphorylates TRIP13 (thyroid hormone receptor interactor 13) on tyrosine 56. Phosphorylated (phospho-)TRIP13 promotes non-homologous end joining (NHEJ) repair to induce radiation resistance. NHEJ is the main repair pathway for radiation-induced DNA damage. Tumors expressing high TRIP13 do not respond to radiation but are sensitive to cetuximab or cetuximab combined with radiation. Suppression of phosphorylation of TRIP13 at Y56 abrogates these effects. These findings show that EGFR-mediated phosphorylation of TRIP13 at Y56 is a vital mechanism of radiation resistance. Notably, TRIP13-pY56 could be used to predict the response to radiation or cetuximab and could be explored as an actionable target.

Squamous cell carcinoma subverts adjacent histologically normal epithelium to promote lateral invasion.

Recurrent and new tumors, attributed in part to lateral invasion, are frequent in squamous cell carcinomas and lead to poor survival. We identified a mechanism by which cancer subverts adjacent histologically normal epithelium to enable small clusters of cancer cells to burrow undetected under adjacent histologically normal epithelium. We show that suppression of DMBT1 within cancer promotes aggressive invasion and metastasis in vivo and is associated with metastasis in patients. Cancer cells via TGFß1 and TNFα also suppress DMBT1 in adjacent histologically normal epithelium, thereby subverting it to promote invasion of a small population of tumor cells. The sufficiency of DMBT1 in this process is demonstrated by significantly higher satellite tumor nests in Dmbt1-/- compared with wild-type mice. Moreover, in patients, invasion of small tumor nests under adjacent histologically normal epithelium is associated with increased risk for recurrence and shorter disease-free survival. This study demonstrates a crucial role of adjacent histologically normal epithelium in invasion and its important role in the tumor microenvironment and opens new possibilities for therapeutic strategies that reduce tumor recurrence.

Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA.

BACKGROUND: Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene's transcriptional start site and thereby regulating its own transcription. OBJECTIVE: In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. METHODS: To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted. RESULTS: Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA. CONCLUSION: In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.

Gut microbial dataset of a foraging tribe from rural West Bengal - insights into unadulterated and transitional microbial abundance.

The human gut microbiome contributes to a broad range of biochemical and metabolic functions that directly or indirectly affect human system. Numerous factors such as age, geographical location, genetic makeup, and individual health status significantly influence the diversity, stability, and relative abundance of the gut microbiome. Of the mentioned factors, geographical location and dietary practices appears to explain a significant portion of microbiome variation. On the other hand tribal people living in geographically isolated areas and dependent on their traditional food sources are considered as having relatively unadulterated gut as their guts are least colonized by Western diet. The Western diet - low in fiber and high in refined sugars - is basically wiping out species of bacteria from our intestines. That's the conclusion Smits (2017) and his team reached after analyzing the Hadza microbiome at one stage of their year long study. The trend was clear: The further away people's diets are from a Western diet, the greater the variety of microbes they tend to have in their guts. And that includes bacteria that are missing from American guts."So whether it's people in Africa, Papua New Guinea or South America, communities that live a traditional lifestyle have common gut microbes - ones that we all lack in the industrialized world. In this work we present a pilot study data of the gut microbiome of an ethnic tribe of West Bengal, India, originating from Dravidian descent - the Savars. These are nomadic tribes and are still dependent on hunting and gathering for their livelihood. We identified a healthy family and have analysed their stool samples for gut microbial profiles.

Biophysical characterization Of Alpers encephalopathy associated mutants of human mitochondrial phenylalanyl-tRNA synthetase.

Mutations in nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mitaaRSs) lead to defects in mitochondrial translation affecting the expression and function of 13 subunits of the respiratory chain complex leading to diverse pathological conditions. Mutations in the FARS2 gene encoding human mitochondrial phenylalanyl-tRNA synthetase (HsmitPheRS) have been found to be associated with two different clinical representations, infantile Alpers encephalopathy and spastic paraplegia. Here we have studied three pathogenic mutants (Tyr144Cys, Ile329Thr, and Asp391Val) associated with Alpers encephalopathy to understand how these variants affect the biophysical properties of the enzyme. These mutants have already been reported to have reduced aminoacylation activity. Our study established that the mutants are significantly more thermolabile compared to the wild-type enzyme with reduced solubility in vitro. The presence of aggregation-prone insoluble HsmitPheRS variants could have a detrimental impact on organellar translation, and potentially impact normal mitochondrial function. © 2019 IUBMB Life, 71(8): 1141-1149, 2019 © 2019 IUBMB Life, 71(8):1141-1149, 2019.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica.

BACKGROUND: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. OBJECTIVE: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. METHODS: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. RESULTS: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. CONCLUSION: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two - state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress.

BACKGROUND: Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis. METHODS: Non-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress on ScmitPheRS activity. RESULTS: ScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress. CONCLUSION: Formation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhe binding of the enzyme, thus inactivating ScmitPheRS reversibly. GENERAL SIGNIFICANCE: The ScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity of ScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress.

p-Benzoquinone-induced aggregation and perturbation of structure and chaperone function of α-crystallin is a causative factor of cigarette smoke-related cataractogenesis.

Cigarette smoking is a significant risk factor for cataract. However, the mechanism by which cigarette smoke (CS) causes cataract remains poorly understood. We had earlier shown that in CS-exposed guinea pig, p-benzoquinone (p-BQ) derived from CS in the lungs is carried by the circulatory system to distant organs and induces various smoke-related pathogeneses. Here, we observed that CS exposure caused accumulation of the p-BQ-protein adduct in the eye lens of guinea pigs. We also observed accumulation of the p-BQ-protein adduct in resected lens from human smokers with cataract. No such accumulation was observed in the lens of never smokers. p-BQ is a strong arylating agent that forms Michael adducts with serum albumin and haemoglobin resulting in alterations of structure and function. A major protein in the mammalian eye lens is αA-crystallin, which is a potent molecular chaperone. αA-crystallin plays a key role in maintaining the integrity and transparency of the lens. SDS-PAGE indicated that p-BQ induced aggregation of αA-crystallin. Various biophysical techniques including UV-vis spectroscopy, fluorescence spectroscopy, FT-IR, bis-ANS titration suggested a perturbation of structure and chaperone function of αA-crystallin upon p-BQ modification. Our results indicate that p-BQ is a causative agent involved in the modification of αA-crystallin and pathogenesis of CS-induced cataract. Our findings would educate public about the impacts of smoking on eye health and help to discourage them from smoking. The study might also help scientists to develop new drugs for the intervention of CS-induced cataract at an early stage.

HNSCC subverts PBMCs to secrete soluble products that promote tumor cell proliferation.

The immune system detects shifts from homeostasis and eliminates altered cells. However, neoplastic cells can modulate the host response to escape immunosurveillance thereby allowing tumor progression. Head and neck squamous cell carcinoma (HNSCC) is one of the most immunosuppressive cancers but its role in co-opting the immune system to actively promote tumor growth has not been investigated. In this study, we investigated the influence of soluble factors secreted by HNSCC and non-neoplastic epithelial cells on proliferation, apoptosis, activation, cytokine gene expression and phenotypic polarization of immune cells of healthy donors. Then, we determined if the immunomodulation caused by HNSCC-derived soluble products leads to immunosubversion by assessing proliferation, migration and survival of tumor cells exposed to soluble products secreted by modulated immune cells or co-cultured with immune cells. Soluble products from HNSCC inhibited proliferation and cytokine expression in PBMCs, activation of T cells, and polarization of CD4+ towards the Th17 phenotype. These changes co-opted the immune cells to favor cell proliferation, survival and migration of HNSCC. This immunosubversion was observed both indirectly with secreted products and with direct cell-to-cell contact. We conclude that HNSCC-derived secreted products create an immunosuppressive environment that facilitates evasion of tumor cells and subverts the immune cells into a pro-tumoral phenotype.

CDH11 inhibits proliferation and invasion in head and neck cancer.

BACKGROUND: In this study, we use a bioinformatics-based strategy to nominate a tumor suppressor gene cadherin-11 (CDH11) and investigate its role in growth and invasion in head and neck squamous cell carcinoma (HNSCC). METHODS: Using the Oncomine™ database to compare HNSCC and normal specimens, CDH11 was nominated as having a role in HNSCC. CDH11 expression in HNSCC was evaluated by immunohistochemistry on a tissue microarray (TMA) and immunoblotting and immunofluorescence of cell lines. The functional impact of CDH11 on proliferation and invasion was evaluated after siRNA-mediated knockdown. RESULTS: In silico analysis suggested that CDH11 is overexpressed in HNSCC compared to normal specimens. HNSCC TMA exhibited a small but significant increase in intensity and proportion of CDH11. By immunoblot analysis, CDH11 was higher in 4/7 HNSCC cell lines compared to normal keratinocytes; CDH11 was highly upregulated in UM-SCC-47 and UM-SCC-74A and detectable in UM-SCC-14A and UM-SCC-29 cell lines. Downregulation of CDH11 in both UM-SCC-29 and UM-SCC-47 using two different siRNAs enhanced proliferation and invasion. CONCLUSION: CDH11 inhibits cell proliferation and invasion of HNSCC. This suggests that CDH11 functions as a tumor suppressor gene in head and neck cancer. Our findings emphasize the importance of verifying in silico findings with functional studies.