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1.
Int J Mol Sci ; 23(23)2022 Dec 06.
Article in English | MEDLINE | ID: mdl-36499754

ABSTRACT

Oncolytic adenoviruses are promising new anticancer agents. To realize their full anticancer potential, they are being engineered to express therapeutic payloads. Tumor suppressor p53 function contributes to oncolytic adenovirus activity. Many cancer cells carry an intact TP53 gene but express p53 inhibitors that compromise p53 function. Therefore, we hypothesized that oncolytic adenoviruses could be made more effective by suppressing p53 inhibitors in selected cancer cells. To investigate this concept, we attenuated the expression of the established p53 inhibitor synoviolin (SYVN1) in A549 lung cancer cells by RNA interference. Silencing SYVN1 inhibited p53 degradation, thereby increasing p53 activity, and promoted adenovirus-induced A549 cell death. Based on these observations, we constructed a new oncolytic adenovirus that expresses a short hairpin RNA against SYVN1. This virus killed A549 cells more effectively in vitro and inhibited A549 xenograft tumor growth in vivo. Surprisingly, increased susceptibility to adenovirus-mediated cell killing by SYVN1 silencing was also observed in A549 TP53 knockout cells. Hence, while the mechanism of SYVN1-mediated inhibition of adenovirus replication is not fully understood, our results clearly show that RNA interference technology can be exploited to design more potent oncolytic adenoviruses.


Subject(s)
Oncolytic Virotherapy , Oncolytic Viruses , Humans , Adenoviridae/physiology , Oncolytic Viruses/genetics , Oncolytic Viruses/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Oncolytic Virotherapy/methods , Virus Replication/genetics , Cell Line, Tumor , Xenograft Model Antitumor Assays , Ubiquitin-Protein Ligases/metabolism
2.
BMC Bioinformatics ; 19(1): 301, 2018 Aug 20.
Article in English | MEDLINE | ID: mdl-30126372

ABSTRACT

BACKGROUND: Reproducibility of hits from independent CRISPR or siRNA screens is poor. This is partly due to data normalization primarily addressing technical variability within independent screens, and not the technical differences between them. RESULTS: We present "rscreenorm", a method that standardizes the functional data ranges between screens using assay controls, and subsequently performs a piecewise-linear normalization to make data distributions across all screens comparable. In simulation studies, rscreenorm reduces false positives. Using two multiple-cell lines siRNA screens, rscreenorm increased reproducibility between 27 and 62% for hits, and up to 5-fold for non-hits. Using publicly available CRISPR-Cas screen data, application of commonly used median centering yields merely 34% of overlapping hits, in contrast with rscreenorm yielding 84% of overlapping hits. Furthermore, rscreenorm yielded at most 8% discordant results, whilst median-centering yielded as much as 55%. CONCLUSIONS: Rscreenorm yields more consistent results and keeps false positive rates under control, improving reproducibility of genetic screens data analysis from multiple cell lines.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Testing/methods , Genomics/methods , RNA, Small Interfering/genetics , Humans , Reproducibility of Results
3.
Hum Gene Ther ; 28(9): 766-780, 2017 09.
Article in English | MEDLINE | ID: mdl-28114818

ABSTRACT

Oncolytic adenoviruses represent a novel class of anticancer agents. Their efficacy in killing cancer cells is variable, suggesting that there is room for improvement. Host miRNAs have been shown to play important roles in susceptibility of cells to replication of different viruses. This study investigated if adenovirus replication in human prostate cancer cells is influenced by host cell miRNA expression. To this end, human miRNA expression in response to adenovirus infection was analyzed, and functional screens for lytic adenovirus replication were performed using synthetic miRNA mimic and inhibitor libraries. Adenovirus infection generally reduced miRNA expression. On top of this nonspecific interference with miRNA biogenesis, a set of miRNAs, including in particular miR-222, was found specifically reduced. Another set of miRNAs was found to promote adenovirus-induced death of prostate cancer cells. In most cases, this did not stimulate adenovirus propagation. The exception was miR-26b. Overexpression of miR-26b inhibited adenovirus-induced NF-κB activation, augmented adenovirus-mediated cell death, increased adenovirus progeny release, and promoted adenovirus propagation and spread in several human prostate cancer cell lines. This suggests that miR-26b is particularly useful to be combined with oncolytic adenovirus for more effective treatment of prostate cancer.


Subject(s)
Adenoviridae/genetics , MicroRNAs/genetics , Prostatic Neoplasms/genetics , A549 Cells , Cell Death/genetics , Cell Line, Tumor , Gene Expression Regulation/genetics , HEK293 Cells , Humans , Male , NF-kappa B/genetics , Oncolytic Virotherapy/methods , Oncolytic Viruses/genetics , Prostatic Neoplasms/virology , Virus Replication/genetics
4.
Oncotarget ; 7(13): 16676-87, 2016 Mar 29.
Article in English | MEDLINE | ID: mdl-26918338

ABSTRACT

The centrosome plays a key role in cancer invasion and metastasis. However, it is unclear how abnormal centrosome numbers are regulated when prostate cancer (PCa) cells become metastatic. CP110 was previously described for its contribution of centrosome amplification (CA) and early development of aggressive cell behaviour. However its regulation in metastatic cells remains unclear. Here we identified miR-129-3p as a novel metastatic microRNA. CP110 was identified as its target protein. In PCa cells that have metastatic capacity, CP110 expression was repressed by miR-129-3p. High miR-129-3p expression levels increased cell invasion, while increasing CP110 levels decreased cell invasion. Overexpression of CP110 in metastatic PCa cells resulted in a decrease in the number of metastasis. In tissues of PCa patients, low CP110 and high miR-129-3p expression levels correlated with metastasis, but not with the expression of genes related to EMT. Furthermore, overexpression of CP110 in metastatic PCa cells resulted in excessive-CA (E-CA), and a change in F-actin distribution which is in agreement with their reduced metastatic capacity. Our data demonstrate that miR-129-3p functions as a CA gatekeeper in metastatic PCa cells by maintaining pro-metastatic centrosome amplification (CA) and preventing anti-metastatic E-CA.


Subject(s)
Cell Cycle Proteins/biosynthesis , Centrosome/pathology , Gene Expression Regulation, Neoplastic/physiology , MicroRNAs/metabolism , Microtubule-Associated Proteins/biosynthesis , Phosphoproteins/biosynthesis , Prostatic Neoplasms/pathology , Animals , Cell Line, Tumor , Humans , Male , MicroRNAs/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Rats
5.
Radiat Oncol ; 10: 55, 2015 Feb 27.
Article in English | MEDLINE | ID: mdl-25888875

ABSTRACT

BACKGROUND: Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. METHODS: We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. RESULTS: On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. CONCLUSIONS: We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid the identification of molecular targets for radiosensitization, thereby contributing to improving the efficacy of radiotherapy.


Subject(s)
Cell Survival/radiation effects , High-Throughput Screening Assays/methods , Prostatic Neoplasms/radiotherapy , Radiation Tolerance/genetics , Automation , Caffeine/pharmacology , Central Nervous System Stimulants/pharmacology , Colony-Forming Units Assay , DNA-Activated Protein Kinase/antagonists & inhibitors , DNA-Activated Protein Kinase/genetics , Genome, Human , Humans , Male , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Small Interfering/genetics , Radiation, Ionizing , Radiation-Sensitizing Agents/pharmacology , Tumor Cells, Cultured
6.
Mol Cancer Ther ; 14(6): 1434-44, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25808837

ABSTRACT

Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non-small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule-associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggesting that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradiation showed that a number of cell-cycle-related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G2-M phase arrest and defects in cytokinesis, which are followed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy.


Subject(s)
Carcinoma, Non-Small-Cell Lung/radiotherapy , Forkhead Transcription Factors/genetics , Genome, Human/genetics , Lung Neoplasms/radiotherapy , Microtubule-Associated Proteins/genetics , Protein Serine-Threonine Kinases/genetics , RNA Interference , Animals , Blotting, Western , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle/genetics , Cell Cycle/radiation effects , Cell Line, Tumor , Down-Regulation/genetics , Down-Regulation/radiation effects , Forkhead Box Protein M1 , Forkhead Transcription Factors/metabolism , Gamma Rays , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/radiation effects , HEK293 Cells , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice, Nude , Microscopy, Confocal , Microtubule-Associated Proteins/metabolism , Phosphorylation/genetics , Phosphorylation/radiation effects , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Survival Analysis , Xenograft Model Antitumor Assays
7.
Cardiovasc Res ; 99(3): 471-82, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23536606

ABSTRACT

AIMS: Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith connected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are associated with distinct localized RhoA activities at the subcellular level. METHODS AND RESULTS: Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility. Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for this protrusive activity and maintenance of barrier restoration. CONCLUSION: Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter-endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re-annealing of endothelial junctions.


Subject(s)
Capillary Permeability/physiology , Endothelial Cells/enzymology , rhoA GTP-Binding Protein/metabolism , Actins/metabolism , Animals , Endothelial Cells/physiology , Fluorescence Resonance Energy Transfer , Human Umbilical Vein Endothelial Cells , Humans , Intercellular Junctions/physiology , Male , Rats , Rats, Wistar , Signal Transduction , Thrombin/metabolism
8.
Nucleosides Nucleotides Nucleic Acids ; 30(12): 1214-22, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22132977

ABSTRACT

Deoxycytidine kinase (dCK) is essential for phosphorylation of natural deoxynucleosides and analogs, such as gemcitabine and cytarabine, two widely used anticancer compounds. Regulation of dCK is complex, including Ser-74 phosphorylation. We hypothesized that dCK could be regulated by two additional mechanisms: micro-RNA (miRNA) and promoter methylation. Methylation-specific PCR (MSP) revealed methylation of the 3' GC box in three out of six cancer cell lines. The 3' GC box is located at the dCK promoter region. The methylation status was related to dCK mRNA expression. TargetScan and miRanda prediction algorithms revealed several possible miRNAs targeting dCK and identified miR-330 (micro-RNA 330) as the one conserved between the human, the chimpanzee, and the rhesus monkey genomes. Expression of miR-330 in various colon and lung cancer cell lines, as measured by QRT-PCR, varied five-fold between samples and correlated with in-vitro gemcitabine resistance (R = 0.82, p = 0.04). Exposure to gemcitabine also appeared to influence miR-330 levels in these cell lines. Furthermore, in our cell line panel, miR-330 expression negatively correlated with dCK mRNA expression (R = 0.74), suggesting a role of miR-330 in post-transcriptional regulation of dCK. In conclusion, the 3' GC box and miR-330 may regulate dCK expression in cancer cells.


Subject(s)
DNA Methylation/genetics , Deoxycytidine Kinase/genetics , Deoxycytidine/analogs & derivatives , MicroRNAs/genetics , Promoter Regions, Genetic/genetics , Base Composition/genetics , Base Sequence , Cell Line, Tumor , DNA Methylation/drug effects , Deoxycytidine/pharmacology , Deoxycytidine Kinase/metabolism , Drug Screening Assays, Antitumor , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitory Concentration 50 , MicroRNAs/metabolism , Molecular Sequence Data , Neoplasms/enzymology , Neoplasms/genetics , Protein Binding/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Gemcitabine
9.
EPMA J ; 1(3): 369-375, 2010 Sep.
Article in English | MEDLINE | ID: mdl-21151487

ABSTRACT

Present cancer treatment strategies are based on the assumption that a therapy may work ("response") or not work ("no-response"). However, the existing evidence suggests that current cancer treatment modalities may also have a cancer-promoting effect in part of the patients. In this paper, some relevant data are reviewed suggesting that surgery, irradiation, chemotherapy and immunotherapy can stimulate tumor growth / metastatic spread and decrease survival of patients in certain subgroups. Thus, results of cancer treatment may be improved by detection and use of biomarkers that correlate with positive or negative therapeutic effects. Small trials based on groups with differing biomarkers rather than large phase III trials may aid the development and efficacy testing of new anticancer drugs. Moreover, ignoring biomarkers that correlate with positive or negative therapeutic effect may not be compatible anymore with the ethical principle "First Do No Harm".

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