MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor alpha IMPLICATIONS FOR CANCER THERAPEUTICS

Estrogen-related receptor (ESRRA) functions as a transcription factor and regulates the expression of several genes, such as WNT11 and OPN. Up-regulation of ESRRA has been reported in several cancers. However, the mechanism underlying its up-regulation is unclear. Furthermore, the reports regarding the role and regulation of ESRRA in oral squamous cell carcinoma (OSCC) are completely lacking. Here, we show that tumor suppressor miR-125a directly binds to the 3UTR of ESRRA and represses its expression. Overexpression of miR-125a in OSCC cells drastically reduced the level of ESRRA, decreased cell proliferation, and increased apoptosis. Conversely, the delivery of an miR-125a inhibitor to these cells drastically increased the level of ESRRA, increased cell proliferation, and decreased apoptosis. miR-125a-mediated down-regulation of ESRRA impaired anchorage-independent colony formation and invasion of OSCC cells. Reduced cell proliferation and increased apoptosis of OSCC cells were dependent on the presence of the 3UTR in ESRRA. The delivery of an miR-125a mimic to OSCC cells resulted in marked regression of xenografts in nude mice, whereas the delivery of an miR-125a inhibitor to OSCC cells resulted in a significant increase of xenografts and abrogated the tumor suppressor function of miR-125a. We observed an inverse correlation between the expression levels of miR-125a and ESRRA in OSCC samples. In summary, up-regulation of ESRRA due to down-regulation of miR-125a is not only a novel mechanism for its up-regulation in OSCC, but decreasing the level of ESRRA by using a synthetic miR-125a mimic may have an important role in therapeutic intervention of OSCC and other cancers

Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation IMPLICATIONS FOR CANCER THERAPEUTICS

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates beta-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3'-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC

Noot bij: HR 31 maart 2017, JOR, 2017, 211 (Faillissement Aldel. Verplichting curatoren tot opruimen afvalstoffen jegens gebruiker opslaglocatie)

Item does not contain fulltext31 maart 201

Intraoperative Nerve Monitoring during Minimally Invasive Esophagectomy and 3-Field Lymphadenectomy: Safety, Efficacy, and Feasibility

Background: The objective of this study was to demonstrate the safety, efficacy, and feasibility of intraoperative monitoring of the recurrent laryngeal nerves during thoracoscopic and robotic 3-field esophagectomy. Methods: This retrospective analysis details our initial experience using intraoperative nerve monitoring (IONM) during minimally invasive 3-field esophagectomy. Data were obtained from a prospectively maintained database and electronic medical records. The study included all patients who underwent minimally invasive (video-assisted thoracic surgery/robotic) transthoracic esophagectomy with neck anastomosis. The patients were divided into those who underwent IONM during the study period and a historical cohort who underwent 3-field esophagectomy without IONM at the same institution. Appropriate statistical tests were used to compare the 2 groups. Results: Twenty-four patients underwent nerve monitoring during minimally invasive 3-field esophagectomy. Of these, 15 patients underwent thoraco-laparoscopic operation, while 9 received a robot-assisted procedure. In the immediate postoperative period, 8 of 24 patients (33.3%) experienced vocal cord paralysis. Relative to a historical cohort from the same institution, who were treated with surgery without nerve monitoring in the preceding 5 years, a 26% reduction was observed in the nerve paralysis rate (p=0.08). On follow-up, 6 of the 8 patients with vocal cord paralysis reported a return to normal vocal function. Additionally, patients who underwent IONM exhibited a higher nodal yield and a decreased frequency of tracheostomy and bronchoscopy. Conclusion: The use of IONM during minimally invasive 3-field esophagectomy is safe and feasible. This technique has the potential to decrease the incidence of recurrent nerve palsy and increase nodal yield

Methylation of CpG islands in the <i>MCPH1</i> promoter.

<p>(A) Schematic representation of the <i>MCPH1</i> promoter with two CpG islands. The vertical lines represent the CpG sites. The solid and open horizontal arrows represent primers to amplify CpGI and CpGII islands respectively. Sites for <i>Bst</i> UI and <i>Aci</i> I in CpGI and CpGII, respectively, are marked by filled vertical arrowheads. The numbers represent nucleotide positions with respect to the TSS. (B) Representative agarose gel images of COBRA for CpGI (upper panel) and CpGII (lower panel). Note the absence of methylation of CpGI in tumor samples 95T and 150T, and the methylation of CpGII in tumor samples 80T and 116T. (C) Schematic representation of bisulfite treated genomic DNA sequence of CpGII in normal and tumor tissues from patient numbers 80, 116, 177 and 202. Each row represents a sequenced TA clone. The filled and unfilled squares represent methylated and unmethylated CpGs respectively. Note the methylation of tumor samples and non-methylation of their corresponding normal oral tissues. (D) Representative agarose gel images of COBRA data for CpGI (upper panel) and CpGII (lower panel) in cell lines. None of the cell lines show CpGI methylation, whereas CpGII shows methylation in SCC084 cells only. (E) Bisulfite sequencing of CpGII in SCC084 cells before and after the AZA (2′-deoxy-5-azacytidines) treatment. The CpG sites in CpGII show methylation in DMSO (vehicle control) treated DNA, whereas, as expected, methylation is lost after AZA treatment. Abbreviations: N, normal; T, tumor; PD, positive control (<i>ASPM</i> fragment); PU, positive control undigested; UN, undigested CpG island I or II; and, NB1 or NB2, peripheral blood DNA from unrelated normal individuals. Numbers represent patient numbers.</p

LOH at the MCPH1 locus.

<p>(A) Representative phosphor images showing LOH for the markers D8S1819, D8S277 and D8S1798 flanking the MCPH1 locus. B & T denote constitutive blood/normal oral tissue and tumor DNA respectively. Arrows indicate the loss of alleles in tumor DNA. (B) Diagrammatic representation of LOH data from 81 matched blood/normal oral tissue and tumor DNA samples using three microsatellite markers. Tumor samples are arranged according to their T classification (T1 to T4) or ED (epithelial dysplasia). Abbreviations: NI, non-informative; IN, informative; MSI, microsatellite instability; and, L, LOH.</p

miR-27a targets both the seed regions of <i>MCPH1</i> cloned separately.

<p><b>(</b>A) The luciferase reporter assay of different constructs harboring seed region 1 (SDR1) of <i>MCPH1</i> 3′-UTR in KB cells. Note a significantly reduced luciferase activity in cells co-transfected with pMIR-Report-3′-UTR-S1 and pcDNA3/pre-miR-27a/<i>EGFP</i> (0.2 or 0.4 µg) in comparison to those with pMIR-Report, suggesting miR-27a targets SDR1 of <i>MCPH1</i>. As expected, no significant difference in luciferase activity was observed in cells co-transfected with pMIR-Report-3′-UTR-AS1 or pMIR-Report-3′-UTR-M1 with pcDNA3/pre-miR-27a/<i>EGFP</i> (0.2 or 0.4 µg) in comparison to those with pMIR-Report. (B) The luciferase reporter assay of different constructs harboring seed region 2 (SDR2) of <i>MCPH1</i> 3′-UTR in KB cells. As with SDR1, miR-27a also targets SDR2.</p

Increased apoptosis and decreased invasion in MCPH1 overexpressing cells.

<p>(A) The analysis of sub-G1 populations (a measure of cell death) in PI stained KB, V2, V4, B1 and B9 cells by flow cytometry. The graph represents one of the three separate experiments. Note higher sub-G1 populations in MCPH1 overexpressing B1 and B9 cells in comparison to KB, V2 and V4 cells. The mean values (%) ± SDs for sub-G1 populations in different cells are as follows: KB, 5.07±0.63; V2, 2.06±0.71; V4, 2.40±0.48; B1, 16.16±0.57; and B9, 22.64±0.45. (B) Analysis of apoptosis by <i>in </i><i>vitro</i> quantitation of CASP3 activity. Note significantly increased CASP3 activity in B1 and B9 cells in comparison to KB, V2 and V4 cells. The values shown are mean±SD of three separate experiments. (C) Analysis of cell invasion in KB cells, and V2, V4, B1 and B9 clones. Note <i>MCPH1</i> overexpression reduced invasiveness in B1 and B9 cells as compared to KB, V2 and V4 cells. (D) The quantitative representation of the cell invasion assay data. The values are the mean±SD of the number of invaded cells counted in four random microscopic fields. Abbreviations: ns, statistically not significant; **p<0.005; and, ***p<0.001.</p