Browsing by Subject "Gene Expression Regulation, Neoplastic"
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Item Open Access Herpes simplex virus 1 amplicon vector-mediated siRNA targeting epidermal growth factor receptor inhibits growth of human glioma cells in vivo(2005) Saydam O.; Glauser, D.L.; Heid I.; Turkeri G.; Hilbe, M.; Jacobs, A.H.; Ackermann, M.; Fraefel, C.In primary glioblastomas and other tumor types, the epidermal growth factor receptor (EGFR) is frequently observed with alterations, such as amplification, structural rearrangements, or overexpression of the gene, suggesting an important role in glial tumorigenesis and progression. In this study, we investigated whether posttranscriptional gene silencing by vector-mediated RNAi to inhibit EGFR expression can reduce the growth of cultured human gli36 glioma cells. To "knock down" EGFR expression, we have created HSV-1-based amplicons that contain the RNA polymerase III-dependent H1 promoter to express double-stranded hairpin RNA directed against EGFR at two different locations (pHSVsiEGFR I and pHSVsiEGFR II). We demonstrate that both pHSVsiEGFR I and pHSVsiEGFR II mediated knock-down of transiently transfected full-length EGFR or endogenous EGFR in a dose-dependent manner. The knock-down of EGFR resulted in the growth inhibition of human glioblastoma (gli36-luc) cells both in culture and in athymic mice in vivo. Cell cycle analysis and annexin V staining revealed that siRNA-mediated suppression of EGFR induced apoptosis. Overall HSV-1 amplicons can mediate efficient and specific posttranscriptional gene silencing. Copyright © The American Society of Gene Therapy.Item Open Access Imetelstat (a telomerase antagonist) exerts off target effects on the cytoskeleton(2013) Mender I.; Senturk, S.; Ozgunes, N.; Can Akcali, K.; Kletsas, D.; Gryaznov, S.; Can, A.; Shay J.W.; Dikmen, Z.G.Telomerase is a cellular ribonucleoprotein reverse transcriptase that plays a crucial role in telomere maintenance. This enzyme is expressed in approximately 90% of human tumors, but not in the majority of normal somatic cells. Imetelstat sodium (GRN163L), is a 13-mer oligonucleotide N3'→P5' thio-phosphoramidate lipid conjugate, which represents the latest generation of telomerase inhibitors targeting the template region of the human functional telomerase RNA (hTR) subunit. In preclinical trials, this compound has been found to inhibit telomerase activity in multiple cancer cell lines, as well as in vivo xenograft mouse models. Currently, GRN163L is being investigated in several clinical trials, including a phase II human non small cell lung cancer clinical trial, in a maintenance setting following standard doublet chemotherapy. In addition to the inhibition of telomerase activity in cancer cell lines, GRN163L causes morphological cell rounding changes, independent of hTR expression or telomere length. This leads to the loss of cell adhesion properties; however, the mechanism underlying this effect is not yet fully understood. In the present study, we observed that GRN163L treatment leads to the loss of adhesion in A549 lung cancer cells, due to decreased E-cadherin expression, leading to the disruption of the cytoskeleton through the alteration of actin, tubulin and intermediate filament organization. Consequently, the less adherent cancer cells initially cease to proliferate and are arrested in the G1 phase of the cell cycle, accompanied by decreased matrix metalloproteinase-2 (MMP-2) expression. These effects of GRN163L are independent of its telomerase catalytic activity and may increase the therapeutic efficacy of GRN163L by decreasing the adhesion, proliferation and metastatic potential of cancer cells in vivo.Item Open Access Metastasis suppressor proteins in cutaneous squamous cell carcinoma(Elsevier, 2016-07) Bozdogan, O.; Vargel, I.; Cavusoglu, T.; Karabulut, A. A.; Karahan, G.; Sayar, N.; Atasoy, P.; Yulug, I. G.Cutaneous squamous cell carcinomas (cSCCs) are common human carcinomas. Despite having metastasizing capacities, they usually show less aggressive progression compared to squamous cell carcinoma (SCC) of other organs. Metastasis suppressor proteins (MSPs) are a group of proteins that control and slow-down the metastatic process. In this study, we established the importance of seven well-defined MSPs including NDRG1, NM23-H1, RhoGDI2, E-cadherin, CD82/KAI1, MKK4, and AKAP12 in cSCCs. Protein expression levels of the selected MSPs were detected in 32 cSCCs, 6 in situ SCCs, and two skin cell lines (HaCaT, A-431) by immunohistochemistry. The results were evaluated semi-quantitatively using the HSCORE system. In addition, mRNA expression levels were detected by qRT-PCR in the cell lines. The HSCOREs of NM23-H1 were similar in cSCCs and normal skin tissues, while RGHOGDI2, E-cadherin and AKAP12 were significantly downregulated in cSCCs compared to normal skin. The levels of MKK4, NDRG1 and CD82 were partially conserved in cSCCs. In stage I SCCs, nuclear staining of NM23-H1 (NM23-H1nuc) was significantly lower than in stage II/III SCCs. Only nuclear staining of MKK4 (MKK4nuc) showed significantly higher scores in in situ carcinomas compared to invasive SCCs. In conclusion, similar to other human tumors, we have demonstrated complex differential expression patterns for the MSPs in in-situ and invasive cSCCs. This complex MSP signature warrants further biological and experimental pathway research.Item Open Access MicroRNA-519a is a novel oncomir conferring tamoxifen resistance by targeting a network of tumour-suppressor genes in ER+ breast cancer(John Wiley and Sons Ltd, 2014) Ward, A.; Shukla, K.; Balwierz, A.; Soons, Z.; König, R.; Sahin, O.; Wiemann, S.Tamoxifen is an endocrine therapy which is administered to up to 70% of all breast cancer patients with oestrogen receptor alpha (ERα) expression. Despite the initial response, most patients eventually acquire resistance to the drug. MicroRNAs (miRNAs) are a class of small non-coding RNAs which have the ability to post-transcriptionally regulate genes. Although the role of a few miRNAs has been described in tamoxifen resistance at the single gene/target level, little is known about how concerted actions of miRNAs targeting biological networks contribute to resistance. Here we identified the miRNA cluster, C19MC, which harbours around 50 mature miRNAs, to be up-regulated in resistant cells, with miRNA-519a being the most highly up-regulated. We could demonstrate that miRNA-519a regulates tamoxifen resistance using gain- and loss-of-function testing. By combining functional enrichment analysis and prediction algorithms, we identified three central tumour-suppressor genes (TSGs) in PI3K signalling and the cell cycle network as direct target genes of miR-519a. Combined expression of these target genes correlated with disease-specific survival in a cohort of tamoxifen-treated patients. We identified miRNA-519a as a novel oncomir in ER+ breast cancer cells as it increased cell viability and cell cycle progression as well as resistance to tamoxifen-induced apoptosis. Finally, we could show that elevated miRNA-519a levels were inversely correlated with the target genes' expression and that higher expression of this miRNA correlated with poorer survival in ER+ breast cancer patients. Hence we have identified miRNA-519a as a novel oncomir, co-regulating a network of TSGs in breast cancer and conferring resistance to tamoxifen. Using inhibitors of such miRNAs may serve as a novel therapeutic approach to combat resistance to therapy as well as proliferation and evasion of apoptosis in breast cancer.Item Open Access miR-200c: a versatile watchdog in cancer progression, EMT, and drug resistance(Springer Verlag, 2016-06) Mutlu, M.; Raza, U.; Saatci, Ö.; Eyüpoğlu, E.; Yurdusev, E.; Şahin, Ö.MicroRNAs (miRNAs) are 20–22-nucleotide small endogenous non-coding RNAs which regulate gene expression at post-transcriptional level. In the last two decades, identification of almost 2600 miRNAs in human and their potential to be modulated opened a new avenue to target almost all hallmarks of cancer. miRNAs have been classified as tumor suppressors or oncogenes depending on the phenotype they induce, the targets they modulate, and the tissue where they function. miR-200c, an illustrious tumor suppressor, is one of the highly studied miRNAs in terms of development, stemness, proliferation, epithelial-mesenchymal transition (EMT), therapy resistance, and metastasis. In this review, we first focus on the regulation of miR-200c expression and its role in regulating EMT in a ZEB1/E-cadherin axis-dependent and ZEB1/E-cadherin axis-independent manner. We then describe the role of miR-200c in therapy resistance in terms of multidrug resistance, chemoresistance, targeted therapy resistance, and radiotherapy resistance in various cancer types. We highlight the importance of miR-200c at the intersection of EMT and chemoresistance. Furthermore, we show how miR-200c coordinates several important signaling cascades such as TGF-β signaling, PI3K/Akt signaling, Notch signaling, VEGF signaling, and NF-κB signaling. Finally, we discuss miR-200c as a potential prognostic/diagnostic biomarker in several diseases, but mainly focusing on cancer and its potential application in future therapeutics.Item Open Access The miR-644a/CTBP1/p53 axis suppresses drug resistance by simultaneous inhibition of cell survival and epithelialmesenchymal transition in breast cancer(Impact Journals LLC, 2016) Raza, U.; Saatci, O.; Uhlmann, S.; Ansari, S. A.; Eyüpoglu, E.; Yurdusev, E.; Mutlu, M.; Ersan, P. G.; Altundağ, M. K.; Zhang, J. D.; Dogan, H. T.; Güler, G.; Şahin, Ö.Tumor cells develop drug resistance which leads to recurrence and distant metastasis. MicroRNAs are key regulators of tumor pathogenesis; however, little is known whether they can sensitize cells and block metastasis simultaneously. Here, we report miR-644a as a novel inhibitor of both cell survival and EMT whereby acting as pleiotropic therapy-sensitizer in breast cancer. We showed that both miR-644a expression and its gene signature are associated with tumor progression and distant metastasis-free survival. Mechanistically, miR-644a directly targets the transcriptional co-repressor C-Terminal Binding Protein 1 (CTBP1) whose knock-outs by the CRISPRCas9 system inhibit tumor growth, metastasis, and drug resistance, mimicking the phenotypes induced by miR-644a. Furthermore, downregulation of CTBP1 by miR-644a upregulates wild type- or mutant-p53 which acts as a 'molecular switch' between G1-arrest and apoptosis by inducing cyclin-dependent kinase inhibitor 1 (p21, CDKN1A, CIP1) or pro-apoptotic phorbol-12-myristate-13-acetate-induced protein 1 (Noxa, PMAIP1), respectively. Interestingly, an increase in mutant-p53 by either overexpression of miR-644a or downregulation of CTBP1 was enough to shift this balance in favor of apoptosis through upregulation of Noxa. Notably, p53- mutant patients, but not p53-wild type ones, with high CTBP1 have a shorter survival suggesting that CTBP1 could be a potential prognostic factor for breast cancer patients with p53 mutations. Overall, re-activation of the miR-644a/CTBP1/p53 axis may represent a new strategy for overcoming both therapy resistance and metastasis.Item Open Access Quantification of SLIT-ROBO transcripts in hepatocellular carcinoma reveals two groups of genes with coordinate expression(BioMed Central, 2008) Avci, M. E.; Konu, O.; Yagci, T.Background: SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of SLIT-ROBO genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified SLIT-ROBO transcripts in HCC cell lines, and in normal and tumor tissues from liver. Methods: Expression of SLIT-ROBO family members was quantified by real-time qRT-PCR in 14 HCC cell lines, 8 normal and 35 tumor tissues from the liver. ANOVA and Pearson's correlation analyses were performed in R environment, and different clinicopathological subgroups were pairwise compared in Minitab. Gene expression matrices of cell lines and tissues were analyzed by Mantel's association test. Results: Genewise hierarchical clustering revealed two subgroups with coordinate expression pattern in both the HCC cell lines and tissues: ROBO1, ROBO2, SLIT1 in one cluster, and ROBO4, SLIT2, SLIT3 in the other, respectively. Moreover, SLIT-ROBO expression predicted AFP-dependent subgrouping of HCC cell lines, but not that of liver tissues. ROBO1 and ROBO2 were significantly up-regulated, whereas SLIT3 was significantly down-regulated in cell lines with high-AFP background. When compared to normal liver tissue, ROBO1 was found to be significantly overexpressed, while ROBO4 was down-regulated in HCC. We also observed that ROBO1 and SLIT2 differentiated histopathological subgroups of liver tissues depending on both tumor staging and differentiation status. However, ROBO4 could discriminate poorly differentiated HCC from other subgroups. Conclusion: The present study is the first in comprehensive and quantitative evaluation of SLIT-ROBO family gene expression in HCC, and suggests that the expression of SLIT-ROBO genes is regulated in hepatocarcinogenesis. Our results implicate that SLIT-ROBO transcription profile is bi-modular in nature, and that each module shows intrinsic variability. We also provide quantitative evidence for potential use of ROBO1, ROBO4 and SLIT2 for prediction of tumor stage and differentiation status.Item Open Access SIP1 is downregulated in hepatocellular carcinoma by promoter hypermethylation(2011) Acun, T.; Oztas, E.; Yagci, T.; Yakicier, M.C.Background: Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas.Methods: SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions.Results: We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases.Conclusions: Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis. © 2011 Acun et al; licensee BioMed Central Ltd.