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Anaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CAE545K into the LAM1 PTC cell line, which carries a BRAFV600E mutation. In culture, the engineered cell line (LAM1:PIK3CAE545K) proliferated faster and demonstrated increased clonogenic potential relative to the parental line carrying an empty vector (LAM1EV). Both the LAM1EV and LAM1:PIK3CAE545K edited lines were implanted into hind flanks of athymic nude mice for in vivo determination of disease progression. While tumour weights and volumes were not significantly higher in LAM1:PIK3CAE545K mice, there was a decrease in expression of thyroid differentiation markers TTF-1, thyroglobulin, PAX8 and B-catenin, suggesting that introduction of PIK3CAE545K led to dedifferentiation in vivo. Collectively, this study provides evidence of a role for PIK3CAE545K in driving disease progression from a well-differentiated to an undifferentiated thyroid cancer; however, over-expression was not a determinant of an accelerated growth phenotype in ATC. Graphical Abstract

Loss of the 3p chromosome arm has previously been reported to be a biomarker of poorer outcome in both human papillomavirus (HPV)-positive and HPV-negative head and neck cancer. However, the precise operational measurement of 3p arm loss is unclear and the mutational profile associated with the event has not been thoroughly characterized. We downloaded the clinical, single nucleotide variation (SNV), copy number aberration (CNA), RNA sequencing, and reverse phase protein assay (RPPA) data from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas HNSCC cohorts. Survival data and hypoxia scores were downloaded from published studies. In addition, we report the inclusion of an independent Memorial Sloan Kettering cohort. We assessed the frequency of loci deletions across the 3p arm separately in HPV-positive and -negative disease. We found that deletions on chromosome 3p were almost exclusively an all or none event in the HPV-negative cohort, patients either had &lt, 1% or &gt, 97% of the arm deleted. 3p arm loss, defined as &gt, 97% deletion in HPV-positive patients and &gt, 50% in HPV-negative patients, had no impact on survival (p &gt, 0.05). However, HPV-negative tumors with 3p arm loss presented at a higher N-category and overall stage and developed more distant metastases (p &lt, 0.05). They were enriched for SNVs in TP53, and depleted for point mutations in CASP8, HRAS, HLA-A, HUWE1, HLA-B, and COL22A1 (false discovery rate, FDR &lt, 0.05). 3p arm loss was associated with CNAs across the whole genome (FDR &lt, 0.1), and pathway analysis revealed low lymphoid–non-lymphoid cell interactions and cytokine signaling (FDR &lt, 0.1). In the tumor microenvironment, 3p arm lost tumors had low immune cell infiltration (FDR &lt, 0.1) and elevated hypoxia (FDR &lt, 0.1). 3p arm lost tumors had lower abundance of proteins phospho-HER3 and ANXA1, and higher abundance of miRNAs hsa-miR-548k and hsa-miR-421, which were all associated with survival. There were no molecular differences by 3p arm status in HPV-positive patients, at least at our statistical power level. 3p arm loss is largely an all or none phenomenon in HPV-negative disease and does not predict poorer survival from the time of diagnosis in TCGA cohort. However, it produces tumors with distinct molecular characteristics and may represent a clinically useful biomarker to guide treatment decisions for HPV-negative patients.

Anaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CA

Phosphoinositide 3‐kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K‐PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K‐activated Akt signaling is well established in HNSCC, the significance of mTORC2‐driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2‐specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.

Simple Summary Squamous cell carcinomas from different parts of the larynx have distinct presentations and prognoses, but the molecular basis for this discrepancy has yet to be characterized. We aimed to determine whether different types of mutations at the DNA, mRNA, and protein levels exist to explain the differential prognoses observed. We found that cancers of the supraglottis had higher overall and smoking-associated genome mutations. Further, supraglottic cancers had a significantly poorer prognosis when other clinical variables and mutational status were controlled for. Different protein pathways were enriched in each subsite: muscle-related in the glottis and neural in the supraglottis. Specific cancer-related proteins were also differentially abundant between the supraglottis and glottis. Our findings may partially explain therapeutic response differences, but further study is required for validation. Abstract Laryngeal squamous cell carcinoma (LSCC) from different subsites have distinct presentations and prognosis. In this study, we carried out a multiomic comparison of LSCC subsites. The Cancer Genome Atlas (TCGA) LSCC cohort was analyzed in the R statistical environment for differences between supraglottic and glottic cancers in single nucleotide variations (SNVs), copy number alterations (CNAs), mRNA abundance, protein abundance, pathway overrepresentation, tumor microenvironment (TME), hypoxia status, and patient outcome. Supraglottic cancers had significantly higher overall and smoking-associated SNV mutational load. Pathway analysis revealed upregulation of muscle related pathways in glottic cancer and neural pathways in supraglottic cancer. Proteins involved in cancer relevant signaling pathways including PI3K/Akt/mTOR, the cell cycle, and PDL1 were differentially abundant between subsites. Glottic and supraglottic tumors have different molecular profiles, which may partially account for differences in presentation and response to therapy.

Head and neck squamous cell carcinoma (HNSCC) is characterized by aggressive behavior with a tendency for recurrence and metastasis. Analyses of The Cancer Genome Atlas (TCGA) data and other cohort studies suggest that the loss of the chromosomal 3p arm is a frequent genetic event observed in both human papillomavirus positive and negative HNSCC. Early molecular analyses (i.e. RFLP, CGH) identified three common regions (3p14.2, 3p21.3 and 3p25) that frequently exhibited loss of genetic material on one arm of the 3p chromosome. More recently, next generation sequencing has revealed the loss of larger regions of this arm. Here we review the role of chromosomal 3p arm loss in early initiation and progression of HNSCC, and its relationship with poor patient prognosis.

Objectives The PI3K/AKT/mTOR pathway is frequently altered in head and neck squamous cell cancer (HNSCC), making this pathway a logical therapeutic target. However, PI3K targeting is not universally effective. Biomarkers of response are needed to stratify patients likely to derive benefit and exclude those unlikely to respond. Materials and methods We examined the sensitivity of cell lines with constitutively-active (G12V mutant) HRAS and wild-type HRAS to PI3K inhibition using flow cytometry and cell viability assays. We then overexpressed and silenced HRAS and measured sensitivity to the PI3K inhibitor BYL719. Immunoblotting was used to determine activation of the PI3K pathway. MEK and mTOR inhibitors were then tested in HRAS mutant cells to determine their efficacy. Results HRAS mutant cell lines were non-responsive to PI3K inhibition. Overexpression of HRAS led to reduced susceptibility to PI3K inhibition, while knockdown improved sensitivity. Immunoblotting revealed suppressed AKT phosphorylation upon PI3K inhibition in both wild-type and HRAS mutant cell lines, however mutant lines maintained phosphorylation of S6, downstream of mTOR. Targeting mTOR effectively reduced viability of HRAS mutant cells and we subsequently examined the ERK-TSC2-mTOR cascade as a mediator of resistance to PI3K inhibition. Conclusions HRAS mutant cells are resistant to PI3K inhibition and our findings suggest the involvement of a signalling intersection of the MAPK and PI3K pathways at the level of ERK-TSC2, leading to persistent mTOR activity. mTOR inhibition alone or in combination with MAPK pathway inhibition may be a promising therapeutic strategy for this subset of HNSCC tumors.

Abtract Background The genomic landscape of head and neck cancer has been reported through The Cancer Genome Atlas project. We attempt to determine if high-risk human papillomavirus (HPV) or frequently mutated genes are correlated with survival in an oral cancer cohort. Methods Patient demographic data along with data from final pathology was collected. Tumor DNA was analyzed using a custom Illumina targeted sequencing panel. Five high-risk HPV types were tested by qPCR. Statistical analyses were used to identify associations between patient outcome and mutational status. Results High-risk HPV types were identified in 7% of cases; HPV status was not associated with survival. Mutations were identified in TP53, TERT promoter, & PIK3CA. Mutations in TP53 were significantly associated with poorer overall survival on multi-variate analysis (p = 0.03). Conclusions Mutations in TP53 were associated with poor patient survival. Expanding our sample size may identify further predictors of outcome to direct customized cancer care.

The anti-tumor properties of cannabinoids have been investigated in many in vitro and in vivo studies. Many of these anti-tumor effects are mediated via cannabinoid receptor types 1 and 2 (CB1 and CB2), comprising the endocannabinoid system (ECS). In this study, we investigated the ECS based on CB 1 and CB 2 receptor gene and protein expression in renal cell carcinoma (RCC) cell lines. In view of their further use for potential treatments, we thus investigated the roles of CB1 and CB2 receptors in the anti-proliferative action and signal transduction triggered by synthetic cannabinoid agonists [such as JWH-133 and WIN 55,212–2 (WIN-55)] in RCC cell lines. Human RCC cell lines were used for this study. The CB 1 and CB 2 gene expression levels were analyzed using real-time PCR. Flow cytometric, immunocytochemical and western blot analyses were performed to confirm CB1 and CB2 receptor protein expression. The anti-proliferative effects of synthetic cannabinoids were investigated on cell viability assay. The CB1 and CB2 receptors were blocked pharmacologically with the antagonists SR141716A and AM-630, respectively, to investigate the effects of the agonists JWH-133 and WIN-55. Cell cycle, apoptosis and LDH-based cytotoxicity were analyzed on cannabinoid-treated RCC cells. The CB1 and CB2 genes expression was shown by real-time PCR and flow cytometric and western blot analysis indicating a higher level of CB2 receptor as compared to CB1 in RCC cells. Immunocytochemical staining also confirmed the expression of the CB1 and CB2 proteins. We also found that the synthetic cannabinoid agonist WIN-55 exerted anti-proliferative and cytotoxic effects by inhibiting the growth of RCC cell lines, while the CB2 agonist JWH-133 did not. Pharmacologically blocking the CB1 and CB2 receptors with their respective antagonists SR141716A and AM-630, followed by the WIN-55 treatment of RCC cells allowed uncovering the involvement of CB2, which led to an arrest in the G0/G1 phase of the cell cycle and apoptosis. This study elucidated the involvement of CB2 in the in vitro inhibition of RCC cells, and future applications of CB2 agonists in the prevention and management of RCC are discussed.

Smoking has historically been recognized as a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). This study aimed to assess the mutational differences between heavy smokers (>20 pack years) and never smokers among the HNSCC patients within The Cancer Genome Atlas (TCGA). Single nucleotide variation and copy number aberration differences between heavy smokers and never smokers were compared within human papillomavirus-positive (HPV-positive) (n = 67) and HPV-negative (n = 431) TCGA cohorts with HNSCC, and the impact of these mutations on survival were assessed. No genes were differentially mutated between smoking and never-smoking patients with HPV-positive tumors. By contrast, in HPV-negative tumors, NSD1 and COL1A11 were found to be more frequently mutated in heavy smokers, while CASP8 was more frequently altered in never smokers. HPV-negative patients with NSD1 mutations experienced significantly improved overall survival compared with NSD1 WT patients. This improved prognosis was validated in an independent cohort of 77 oral cavity cancer patients and a meta-analysis that included 2 additional data sets (688 total patients, hazard ratio for death 0.44, 95% CI, 0.30-0.65). NSD1 mutations are more common in HPV-negative heavy smokers, define a cohort with favorable prognosis, and may represent a clinically useful biomarker to guide treatment deintensification for HPV-negative patients.

Background: Numerous studies have demonstrated poorer outcomes by race/ethnicity in head and neck squamous cell carcinoma (HNSCC). Although some studies have identified differences in socioeconomic status and access to care as important factors affecting outcomes, differences in the genomic and extracellular composition of tumors from patients of different races/ethnicities have yet to be explored. Methods: We downloaded the clinical information, single nucleotide variation (SNV), copy number aberration (CNA), mRNA sequencing, and reverse phase protein assay (RPPA) data from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Altas HNSCC cohorts. Survival data and hypoxia scores were downloaded from published studies. We stratified the cohort into combined racio-ethnic groups (REG) as follows: White/Non-Hispanic (White), Hispanic/Latino (Hispanic), Black/African American (Black), Asian, American Indian/Non-Hispanic (Indigenous American). Cases positive for human papillomavirus (HPV) occurred almost exclusively among White patients (68/71) and thus they were excluded. Results: The HPV-negative cohort contained 354 White, 43 Black, 22 Hispanic and 11 Asian and 1 Indigenous American patient. Black patients had poorer overall and progression-free survival than White patients on univariate and multivariate analysis, respectively (p Citation Format: Hugh A.J. Kim, Peter Y.F. Zeng, Alana Sorgini, Mushfiq H. Shaikh, Neil Mundi, Halema Khan, Danielle MacNeil, Mohammed I. Khan, Krupal Patel, Adrian Mendez, John Yoo, Kevin Fung, Pencilla Lang, David A. Palma, Joe S. Mymryk, John W. Barrett, Paul C. Boutros, Anthony C. Nichols. Multi-omic disparities in head and neck squamous cell carcinomas in patients of different racio-ethnic backgrounds [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR11.

In recent years, genome-wide RNA expression analysis has become a routine tool that offers a great opportunity to study and understand the key role of genes that contribute to carcinogenesis. Various microarray platforms and statistical approaches can be used to identify genes that might serve as prognostic biomarkers and be developed as antitumor therapies in the future. Metastatic renal cell carcinoma (mRCC) is a serious, life-threatening disease, and there are few treatment options for patients. In this study, we performed one-color microarray gene expression (4×44K) analysis of the mRCC cell line Caki-1 and the healthy kidney cell line ASE-5063. A total of 1,921 genes were differentially expressed in the Caki-1 cell line (1,023 upregulated and 898 downregulated). Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) approaches were used to analyze the differential-expression data. The objective of this research was to identify complex biological changes that occur during metastatic development using Caki-1 as a model mRCC cell line. Our data suggest that there are multiple deregulated pathways associated with metastatic clear cell renal cell carcinoma (mccRCC), including integrin-linked kinase (ILK) signaling, leukocyte extravasation signaling, IGF-I signaling, CXCR4 signaling, and phosphoinositol 3-kinase/AKT/mammalian target of rapamycin signaling. The IPA upstream analysis predicted top transcriptional regulators that are either activated or inhibited, such as estrogen receptors, TP53, KDM5B, SPDEF, and CDKN1A. The GSEA approach was used to further confirm enriched pathway data following IPA.

The endocannabinoid system (ECS) is a group of neuromodulatory lipids and their receptors, which are widely distributed in mammalian tissues. ECS regulates various cardiovascular, nervous, and immune system functions inside cells. In recent years, there has been a growing body of evidence for the use of synthetic and natural cannabinoids as potential anticancer agents. For instance, the CB1 and CB2 receptors are assumed to play an important role inside the endocannabinoid system. These receptors are abundantly expressed in the brain and fatty tissue of the human body. Despite recent developments in molecular biology, there is still a lack of knowledge about the distribution of CB1 and CB2 receptors in the human kidney and their role in kidney cancer. To address this gap, we explore and demonstrate the role of the endocannabinoid system in renal cell carcinoma (RCC). In this brief overview, we elucidate the therapeutic aspects of the endocannabinoid system for various cancers and explain how this system can be used for treating kidney cancer. Overall, this review provides new insights into cannabinoids' mechanisms of action in both in vivo and in vitro models, and focuses on recent discoveries in the field.

Inducible nitric oxide synthase (iNOS) expression is associated with the pathogenesis of osteoarthritis (OA). This study was undertaken to investigate whether interleukin-1β (IL-1β)-mediated induction of iNOS can be regulated by microRNA-26a-5p (hsa-miR-26a-5p) in OA. Bioinformatics approaches show that 3'UTR of iNOS mRNA contained the 'seed-matched-sequence' for hsa-miR-26a-5p. IL-1β-induced expression of iNOS correlated with the down-regulation of miR-26a-5p in human OA chondrocytes. hsa-miR-26a-5p directly suppressed the luciferase activity of 3'UTR-iNOS reporter clone. Transfection with pre-miR-26a-5p induced marked silencing of iNOS expression. Activation of NF-κB pathway down-regulated the expression of hsa-miR-26a-5p and induced iNOS expression. In short, this is the first report that shows hsa-miR-26a-5p is a direct regulator of iNOS expression in human chondrocytes. hsa-miR-26a-5p may be an important regulator of human cartilage homeostasis and a new target for OA therapy.

Objectives Survival in head and neck squamous cell carcinoma (HNSCC) has been associated with patient sex, typically with males experiencing poorer outcomes. It is unclear if this disparity is based in divergent tumor biology. We analyzed the TCGA HNSCC cohort to uncover disparities in the somatic single nucleotide variation (SNV), copy number alteration (CNA) and mRNA abundance profiles between males and females. Critically, we stratified our results by tumor HPV status to control for this significant confounder. Methods SNV, CNA and mRNA abundance differences between males and females were compared separately for the HPV-positive (n = 67) and negative (n = 431) TCGA HNSCC cohorts. Overall survival outcomes were compared in males and females in both HPV-positive and HPV-negative subsets of patients. Results Females were found to have poorer overall survival than males (p = 0.048), largely due to higher rates of HPV-positive disease among men. SNV analysis revealed that in HPV-positive disease, there were no differences by sex after accounting for the false discovery rate (FDR). In HPV-negative tumors, BRWD3 mutations occurred more frequently in the tumors of female patients compared to males after adjusting for the FDR (p = 0.02). Further, HPV-negative BRWD3 mutant tumors were found to have significantly worse 5-year overall survival compared to wildtype on multivariate analysis (p = 0.02). There were 88 heterozygous deletions and 14 amplifications that were differentially altered between male and female HPV-negative tumors and associated with expression changes. Pathway analysis of these genes revealed that tumors from males were enriched in five pathways including chemokine and phosphophatidylinositol signaling. Conclusions Reanalysis of the TCGA HNSCC dataset stratified by sex revealed that males in this cohort had a significant survival advantage, due to a higher proportion of HPV-positive disease. Mutations in BRWD3 were more frequent in HPV-negative tumors of females and were associated with poorer overall survival. BRWD3 may represent a novel biomarker of patient outcomes, but will require additional validation.

The increase in the life expectancy of patients with renal cell carcinoma (RCC) in the last decade is due to changes that have occurred in the area of preclinical studies. Understanding cancer pathophysiology and the emergence of new therapeutic options, including immunotherapy, would not be possible without proper research. Before new approaches to disease treatment are developed and introduced into clinical practice they must be preceded by preclinical tests, in which animal studies play a significant role. This review describes the progress in animal model development in kidney cancer research starting from the oldest syngeneic or chemically-induced models, through genetically modified mice, finally to xenograft, especially patient-derived, avatar and humanized mouse models. As there are a number of subtypes of RCC, our aim is to help to choose the right animal model for a particular kidney cancer subtype. The data on genetic backgrounds, biochemical parameters, histology, different stages of carcinogenesis and metastasis in various animal models of RCC as well as their translational relevance are summarized. Moreover, we shed some light on imaging methods, which can help define tumor microstructure, assist in the analysis of its metabolic changes and track metastasis development.

Purpose Dental arch dimensions are important not only in dentistry (e.g. orthodontists and prosthodontists, and forensic odontology), but also other medical fields, biology, biometrics, painting or sculpture. This study aimed to determine these dimensions in Vietnamese children and compare these measurements across four ethnic groups and genders. Methods A cross-sectional study was conducted on 3204 Vietnamese children at 7 years of age from four major ethnic groups in Vietnam (Kinh, Tay, Thai and Muong). Results The means variables in study subjects were 33.72 ± 2.16 mm for upper inter-canine width (UCW); 52.74 ± 2.55 mm for upper inter-molar width (UMW); 8.69 ± 1.79 mm for upper anterior length (UAL); 29.59 ± 1.97 mm for upper posterior length (UPL); 26.94 ± 2.49 mm for lower inter-canine width (LCW); 45.89 ± 2.59 mm for lower inter-molar width (LMW); 5.04 ± 1.53 mm for lower anterior length (LAL); and 26.22 ± 2.07 mm for lower posterior length (LPL). The UCM, UMW, and LMW of Muong were significantly wider in males, but narrower in females compared with other ethnic groups. The Kinh, Tay and Thai groups had no significant differences between genders in all dimensions, but these sizes were significantly larger in males than females of Muong group. Conclusions This study presents the means of dental arch dimensions in 7 year-old Vietnamese children, and there is no statistical differences in these dimensions between genders of almost studied groups, except Muong group. Ethnic differences are observed only in UCW, UMW and LMW of Muong vs other groups. Furthermore, Vietnamese children have dental arch width similar to the African and Caucasian.

The aim of this study was to compare effect of everolimus on growth of different renal cell carcinoma (RCC) populations and develop experimental design to measure the early response of everolimus in clear cell RCC (ccRCC) cell lines including renal cancer stem cells. Effect of everolimus on RCC cell lines which include primary (786-0) and metastatic (ACHN) RCC cell lines as well as heterogenous populations of tumor cells of different histological RCC subtypes (clear cell RCC and papillary RCC) was measured when treated with everolimus in the range of 1–9 µM. Gene expression profiling using microarray was performed to determine the early response to everolimus in ccRCC cell lines after optimizing concentration of drug. Gene Set Enrichment Analysis (GSEA) was done which mainly focused on basic genes related to mTOR, hormonal and metabolic pathways. Everolimus acts on RCC cells in a dose—dependent manner. In all examined cell lines IC50 dose was possible to calculate after the third day of treatment. In ccRCC lines (parental and stem cell) everolimus changes expression of mTOR complexes elements and elements of related pathways when treated with optimized doses of drug. Characteristic expression profile for ccRCC cells at an early exposure time to everolimus is to elucidate. Wevarie include some basic observations derived from data analysis in the context of mechanism of action of drug with a view to better understand biology of renal cancer cells.

The median survival rate of patients with metastatic renal carcinoma is approximately 10 to 12 months, with up to 50% of patients developing metastases in the lung parenchyma. The molecular basis for metastatic development remains unclear. In the present study, we used renal cell carcinoma (RCC) cells and bronchial epithelial cells, representing metastasis target organ cells, conditioned medium and co-culture models to identify specific gene expression changes responsible for cancer cell viability in a metastatic microenvironment. RCC cell proliferation and migration increased when the culture was supplemented with conditioned medium from lung fibroblasts or pleural epithelial cells. Healthy epithelial cells were, in turn, also stimulated with conditioned medium from RCC cell lines. The mitogen-activated protein kinase (MAPK), interleukin (IL)-6, and phosphatidylinositol 4,5-bisphosphate (PIP2) signaling pathways were identified as deregulated upon cell‑cell interaction. Thus, cell-cell communication may contribute to the development of the metastatic niche. The identified deregulated signaling pathways may be considered as potential therapeutic targets in metastatic renal carcinoma.

Medicinal plants play a crucial role in maintaining human health. Kalanchoe pinnata has been used for treatment of different ailments like infections, rheumatism, and inflammation. Data showed that Kalancho pinnata methanolic extract (100 or 1000 μg/ml) significantly inhibited growth of shoots (hypocotyls) and roots (radicals) of rice compared to control after 3 or 7 days treatment. Further, the Kalancho pinnata methanolic extract (50–1000 μg/ml) exhibited antioxidant properties and scavenged free radicals in dose-dependent manner compared with a standard antioxidant (ascorbic acid). Kalancho pinnata methanolic extract also possesses antifungal property that inhibited approximately 76% and 51% growth of Aspergillus flavous and Aspergillus niger, respectively, using the extract at 200 μg/ml.

Invasive salmonellosis causes a spectrum of diseases, including enteric fever (i.e., typhoid and paratyphoid fever) and nontyphoidal Salmonella (NTS) infection (1). This group of bacterial pathogens continues to inflict a large burden of disease globally, especially in countries with low and middle incomes (2). No global control strategy yet exists because of multiple challenges, such as low availability of effective vaccines, poor access to clean water and sanitation, lack of regional or national data, and difficulties in scaling up interventions. Recent progress in vaccine development, technology transfer, and disease surveillance has increased prospects of typhoid fever control and eventual control of paratyphoid and NTS.

Background Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells—stem cell-like cancer cells (SCLCCs)—which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Materials and Methods Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers—CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent’s human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Results Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells. Conclusions All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design.

The role of oxidized immunoglobulin G in type 1 diabetic smokers has been investigated in the present study. Human immunoglobulin G (IgG) was modified by reactive oxygen species (ROS). The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified IgG were assessed by direct binding ELISA. High degree of specific binding by 68.5% of patients sera towards ROS-modified IgG was observed in comparison to its native analogue (p< 0.05). In addition, diabetic smokers (n= 28) were examined and the results were compared with diabetic non-smokers (n= 26). Circulating antibodies of diabetic smokers showed substantially stronger binding to modified IgG as compared with the antibodies present in diabetic non-smokers (p< 0.05). Normal human sera (n= 53) showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. The increase in total serum protein carbonyl levels in the diabetic smokers was largely due to an increase in oxidized IgG. Diabetic smokers showed substantially higher carbonyl contents in sera as well as in purified IgG as compared with sera and IgG of diabetic non-smokers. Collectively, the oxidation of plasma proteins, especially IgG, might enhance oxidative stress in type 1 diabetes smokers.

e16091Background: Tumor initiating cells represent a minor cell subpopulation that is resistant to therapies and has cancer stem cell characteristics including self-renewal, multiple lineage differ...

e15600 Background: Cancer cell drug resistance is regulated by arising molecular and biochemical abnormalities in cancer cell on one hand and by tumor micro-environmental factors on the other. A co...

e15577 Background: Cancer cell proliferation is regulated by its genomic and epigenetic abnormalities on one hand and by tumor microenvironment on the other. A complex network of different cell-cel...

488 Background: Tumor microenvironment is a complex network of multiple cell types, paracrine and endocrine signaling molecules, ECM and physical factors, which orchestrate the fate of tumor progression. In the field of ccRCC little attention has been paid to non-immunologic factors in tumor niche. Objectives of this study include the analysis of hypoxia, triiodothyronine (T3), insulin, insulin-like growth factor-1 and -2 impact on ccRCC cells. The effect of above mentioned tumor niche factors on TKI activity on ccRCC cells was to be quantified. The study was initiated as ccRCC cells are known to harbor disrupted hypoxia response and TRbeta (TRβ) - T3 receptor - functions as a tumor suppressor with tissue hypothyroidism involved in maintaining a proliferative advantage, whereas hypothyroidisms was defined as biomarker of sunitinib treatment efficacy. Moreover obesity and diabetes (DM2) are considered risk factors for ccRCC and high IGF-I expression is associated with poor long-term patient survival. This research aims to find molecular background for clinically relevant observations. Methods: ccRCC cells from primary and metastatic tumors (ATCC, MSK collections) along with cancer progenitor cells (CD133+) were cultured in normoxic or hypoxic conditions; in defined media supplemented with T3, IN, IGF, TRβ inhibitor and TKIs (sunitinib, axitinib). Cell proliferation was evaluated using AlamarBlue and MTT. Colony formation potential was evaluated. TRβ gene expression was measured with RealTime-RT PCR and sequenced under standard protocol. Normal kidney proximal tube cells were used as negative control. Results: ccRCC TKI responsiveness is limited by hypoxia, at the same time tumor cell proliferation may be promoted by T3, IN and IGF. Colony formation potential of ccRCC cells under TKI treatment in hypoxic conditions is downregulated. Conclusions: Multiple micro-environmental factors modify the proliferation and/or differentiation of ccRCC cell populations. Understanding the complex molecular feedback loops between cancer cells and the surrounding microenvironment should aid the identification of novel targets for improving TKIs clinical management.

In recent years, cancer stem cells (CSCs)/tumor initiating cells (TICs) have been identified inside different tumors. However, currently used anti-cancer therapies are mostly directed against somatic tumor cells without targeting CSCs/TICs. CSCs/TICs also gain resistance to chemotherapies/radiotherapies. For the development of efficient treatment strategies, choosing the best method for isolation and characterization of CSCs/TICs is still debated among the scientific community. In this review, we summarize recent data concerning isolation techniques for CSCs using magnetic cell sorting and flow cytometry. The review focuses on the strategies for sample preparation during flow cytometric analysis, elaborating biomarkers such as CXCR4, CD105, and CD133. In addition, functional properties characteristic of CSCs/TICs using side population selection through Hoechst 33342 dye, aldehyde dehydrogenase 1, dye-cycle violet, and rhodamine 123 are also discussed. We also include a special focus on enriching CSCs/TICs using three-dimensional cell culture models such as agarose–agarose microbeads and sphere formation.

Metastasis is a complex process that propagates cells from the primary or initial site of the cancer occurrence to distant parts of the body. Cancer cells break from the cancer site and circulate through the bloodstream or lymph vessels, allowing them to reach nearly all parts of the body. These circulating tumour cells (CTCs) contain specialized metastasis-initiating cells (MICs) that reside in the biological heterogeneous primary tumour. Researchers have hypothesized that metastasis of renal cell carcinoma is initiated by circulation of MICs in patients' blood and bone marrow. Based on the cancer stem/progenitor cell concept of carcinogenesis, understanding the molecular phenotypes of metastasis-initiating cells (MICs) in renal cancer could play a vital role in developing strategies for therapeutic interventions in renal cancer. Existence of MICs among CTCs in renal carcinoma has not been proven in large scale. However, some studies have reported that specialized markers are found on the surface of circulating cells from the primary tumour. In mice, MICs have been isolated from CTCs using such markers, which have then been transplanted into xenograft model to show whether they give rise to metastasis in different organs. Considering these findings, in this review we have attempted to summarize the studies connected with MICs and their gene expression profiles that are responsible for metastasis in renal cancer.