Assessing Various Control Samples for Microarray Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma

Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: "malignancy", which separated controls from malignant samples and "cell culture-microenvironment" which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples.</p

Laryngeal squamous cell carcinoma cell lines show high tolerance for siRNA-mediated CDK1 knockdown

Alterations of the cell cycle checkpoints lead to uncontrolled cell growth and result in tumorigenesis. One of the genes essential for cell proliferation and cell cycle regulation is CDK1. This makes it a potential target in cancer therapy. In our previous study we have shown upregulation of this gene in laryngeal squamous cell carcinoma (LSCC). Here we analyze the impact of siRNA-mediated CDK1 knockdown on cell proliferation and viability, measured with cell growth monitoring and colorimetric test (CCK8 assay), respectively. We proved that a reduction of CDK1 expression by more than 50% has no effect on these cellular processes in LSCC cell lines (n=2). Moreover, using microarrays, we analyzed global gene expression deregulation in these cell lines after CDK1 knockdown. We searched for enriched ontologies in the group of identified 137 differentially expressed genes (>2-fold change). Within this group we found 3 enriched pathways: protein binding (GO:0005515), mitotic nuclear division (GO:0007067) and transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169) and a group of 11 genes encoding proteins for which interaction with CDK1 was indicated with the use of bioinformatic tools. Among these genes we propose three: CDK6, CALD1 and FYN as potentially dependent on CDK1

Loss of the MAF Transcription Factor in Laryngeal Squamous Cell Carcinoma

MAF is a transcription factor that may act either as a tumor suppressor or as an oncogene, depending on cell type. We have shown previously that the overexpressed miR-1290 influences MAF protein levels in LSCC (laryngeal squamous cell carcinoma) cell lines. In this study, we shed further light on the interaction between miR-1290 and MAF, as well as on cellular MAF protein localization in LSCC. We confirmed the direct interaction between miR-1290 and MAF 3'UTR by a dual-luciferase reporter assay. In addition, we used immunohistochemistry staining to analyze MAF protein distribution and observed loss of MAF nuclear expression in 58% LSCC samples, of which 10% showed complete absence of MAF, compared to nuclear and cytoplasmatic expression in 100% normal mucosa. Using TCGA data, bisulfite pyrosequencing and CNV analysis, we excluded the possibility that loss-of-function mutations, promoter region DNA methylation or CNV are responsible for MAF loss in LSCC. Finally, we identified genes involved in the regulation of apoptosis harboring the MAF binding motif in their promoter region by applied FIMO and DAVID GO analysis. Our results highlight the role of miR-1290 in suppressing MAF expression in LSCC. Furthermore, MAF loss or mislocalization in FFPE LSCC tumor samples might suggest that MAF acts as a LSCC tumor suppressor by regulating apoptosis.</p

Assessing Various Control Samples for Microarray Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma

Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: “malignancy”, which separated controls from malignant samples and “cell culture-microenvironment” which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples

Global miRNA Expression Profiling Identifies miR-1290 as Novel Potential oncomiR in Laryngeal Carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is the most common group among head and neck cancers. LSCC is characterized by a high incidence in Europe. With the aim of better understanding its genetic background we performed global miRNA expression profiling of LSCC cell lines and primary specimens. By this approach we identified a cohort of 33 upregulated and 9 downregulated miRNA genes in LSCC as compared to epithelial no tumor controls.Within this group we identified overexpression of the novel miR-1290 gene not reported in the context of LSCC before. Using a combined bioinformatical approach in connection with functional analysis we delineated two putative target genes of miR-1290 namely ITPR2 and MAF which are significantly downregulated in LSCC. They are interesting candidates for tumor suppressor genes as they are implicated in apoptosis and other processes deregulated in cancer.Taken together, we propose miR-1290 as the new oncomiR involved in LSCC pathogenesis. Additionally, we suggest that the oncogenic potential of miR-1290 might be expressed by the involvement in downregulation of its target genes MAF and ITPR2

Expression level of miR-1290 target genes.

<p>A) Changes in expression level of <i>MAF</i>, <i>ITPR2</i> and <i>KIF13B</i> genes in UT-SCC-34 treated by miR-1290 inhibitor (black graph) compared to both UT-SCC-34 treated by negative control (dark gray graph) as well as UT-SCC-34 wild type (gray graph). * fold change in UT-SCC-34 treated by miR-1290 inhibitor compared to UT-SCC-34 treated by negative control. B) Changes in expression level of MAF protein in UT-SCC-34 and UT-SCC-107cell lines treated by miR-1290 inhibitor (black graph) compared to both cells treated by negative control (dark gray graph) as well as wild type cells (gray graph). * fold change in cells treated by miR-1290 inhibitor compared to cells treated by negative control.</p

Expression level of selected miRNA and selection scheme of miR-1290 target genes.

<p>A) Overexpression of miR-1290 and miR-1246 in primary LSCC cases (LNA real-time qPCR). B) Schematic presentation of the selection process of miR-1290 candidate genes. C) Expression level of <i>MAF</i>, <i>ITPR2</i> and <i>RGS5</i> in LSCC cell lines and no tumor controls (Affymetrix U133 plus 2.0) based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144924#pone.0144924.ref020" target="_blank">20</a>]. * fold change in LSCC cell lines compared to no tumor controls. D) Expression level of <i>MAF</i> and <i>ITPR2</i> in 22 primary tumor samples compared to 5 no tumor controls.</p