Upregulation of miR-21 in Cisplatin Resistant Ovarian Cancer via JNK-1/c-Jun Pathway

<div><p>Cisplatin has been the most accepted drug for the treatment of ovarian cancer for almost 40 years. Although the majority of patients with ovarian cancer respond to front-line platinum combination chemotherapy, many patients will develop cisplatin-resistance disease, which is extremely rapid and fatal. Although various mechanisms of cisplatin resistance have been postulated, the key molecules involved in such resistance have not been identified. MiRNAs are endogenously expressed small non-coding RNAs, which are evolutionarily conserved and function as post-transcriptional regulators of gene expression. Dysregulation of miRNAs have been associated with cancer initiation, progression and drug resistance. The oncogenic miRNA-21, one of the best-studied miRNAs, is upregulated in almost all human cancers. However, the regulation of miR-21 in cisplatin resistant ovarian cancer cells has not been assessed. In this study, we measured the miR-21 expression by real-time PCR and found upregulation of miR-21 in cisplatin resistant compared with cisplatin sensitive ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated the association of the c-Jun transcription factor to the pri-mir-21 DNA promoter regions. Blocking the JNK-1, the major activator of c-Jun phosphorylation, reduced the expression of pre-mir-21 and increased the expression of its well-known target gene, PDCD4. Overexpression of miR-21 in cisplatin sensitive cells decreased PDCD4 levels and increased cell proliferation. Finally, targeting miR-21 reduced cell growth, proliferation and invasion of cisplatin resistant ovarian cancer cells. These results suggest that the JNK-1/c-Jun/miR-21 pathway contributes to the cisplatin resistance of ovarian cancer cells and demonstrated that miR-21 is a plausible target to overcome cisplatin resistance.</p></div

Effect of miR-21 inhibition in cell growth, proliferation and invasion.

<p>(<b>A</b>) A2780CP20 cells were transiently transfected with a miR-21 antagomir or with a negative antagomir (-) as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. Eight and 24 hours after transfection cells were collected and RNA (including miRNAs) was isolated as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. (<b>B</b>) MiR-21 inhibition reduced cell growth compared to untreated cells (NT) or with the negative control inhibitor (NC-Inh). (<b>C</b>) A2780CP20 cells were transfected as in 5A. A thousand cells were seeded in Petri dishes. Ten days later, the colonies were stained and counted. Inhibition of miR-21 decreased the ability of cells to undergo unlimited division compared with the NC-Inh. (<b>D</b>) Cell invasion was carried out as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. The number of invaded cells was expressed in percentages, taken the NC-Inh as 100%. *p<0.05, **p<0.01 compared to NC-Inh. Columns represent the means of triplicates ± S.E.M.</p

Chromatin immunoprecipitation assay (ChIP).

<p>ChIP assay was performed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. (<b>A</b>) SYBR-I-based real-time PCR amplification of the region containing the c-Jun recognition sequence in the pri-miR-21 DNA. The phospho-c-Jun levels bound to the pri-miR-21 promoter was higher in A2780CP20 cells compared with A2780 cells. (<b>B</b>) SYBR-I-based real-time PCR amplification of a DNA region far of the pre-mir-21 promoter was performed as a control. *p<0.05 compared to control. Columns represent the means of triplicates ± S.E.M.</p

Effect of pre-mir-21 overexpression in A2780 cells.

<p>(<b>A</b>) A2780 cells were stably transfected with pCMV-miR21 or empty pCMV-EV vectors. The miR-21 expression was quantified by qRT-PCR. (<b>B</b>) Western blot analysis shows a decreased expression of PDCD4 levels in A2780-miR-21 compared with A2780-EV cells. (<b>C</b>) Overexpression of miR21 increased cell proliferation (13%, ***p<0.001) in A2780-miR-21 compared with A2780-EV cells. (<b>D</b>) A2780-miR-21 overexpressed clones were more resistant to cisplatin-induced cell death compared with untransfected A2780 cells or with the A2780-EV cells. **p<0.01. Columns represent the means of triplicates ± S.E.M.</p

Effect of JNK-1 inhibition in miR-21 and PDCD4 expression.

<p>A2780CP20 cells were treated with 10 µM SP600125. (<b>A</b>) Western blot shown the inhibition of p-c-Jun after treatment with SP600125 in A2780CP20 cells compared to control (DMSO). (<b>B</b>) SYBR-I-based real-time PCR was performed to calculate the relative pre-mir-21 expression in A2780CP20 cells after treatment with SP600125 inhibitor. (<b>C</b>) Western blot and densitometric analysis of PDCD4 protein expression levels after treatment of A2780CP20 cells with SP600125. (<b>D</b>) PDCD4 protein expression levels after transfection of A2780CP20 with miR-21 oligonucleotide inhibitor. (<b>E</b>) A2780 CP20 cells were transiently transfected with two c-Jun-targeted siRNAs as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section. Western blot analysis shows that both c-Jun-siRNAs decreased the c-Jun levels. SYBR-I-based real-time PCR was performed (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Materials and Methods</a>” section) to calculate the relative pre-mir-21 expression levels in A2780CP20 cells after siRNA-mediated c-Jun silencing. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.</p

RT-PCR and western blot analysis of miRNA-21-related molecules.

<p>(<b>A</b>) Validation of microarrays by RT-PCR. (<b>B</b>) MiR-21 levels in a panel of ovarian cancer cells. MiR-21 levels were expressed relative to the A2780 cells miR-21 levels. IC50s were calculated after 72-hr treatment of cells with different concentrations of cisplatin as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097094#s2" target="_blank">Material and Methods</a>” section. (<b>C</b>) Evaluation of c-Jun and p-c-Jun protein expression in A2780 and A2780CP20 cells. (<b>D</b>) Protein expression analysis of MAPKs in total and nuclear fractions of A2780 and A2780CP20 cells. Expression level in Figures A, C and D are without cisplatin treatment. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.</p

Molecular Targeted Enhanced Ultrasound Imaging of Flk1 Reveals Diagnosis and Prognosis Potential in a Genetically Engineered Mouse Prostate Cancer Model

Molecular imaging techniques used to detect the initiation of disease have the potential to provide the best opportunity for early treatment and cure. This report aimed at testing the possibility that Flk1+ (vascular endothelial growth factor receptor 2), a crucial angiogenesis factor of most tumor cells, could be a molecular targeted imaging marker for the diagnosis and prognosis of cancer. We performed Flk1-targeted microbubble-enhanced ultrasound (US) imaging of prostate cancer in a genetically engineered mouse model with normal-appearing intact US (negative) prostates and with three different tumor sizes (small, medium, and large). Higher levels of Flk1+ molecular signals were identified in the intact US (negative) prostate group by US-targeted imaging and immunohistochemical analysis. The increase in Flk1+ expression occurred prior to the angiogenesis switch-on phase and vascularity peak. After this peak accumulation stage of Flk1+ molecules, lower and stabilized levels of Flk1+ signals were maintained together with tumor growth from small, to medium, to large size. In a longitudinal observation in a subset (n = 5) of mice with established tumors, elevated Flk1+ signals were observed in tissues surrounding the prostate cancer, for example, the ipsilateral boundary zones between two developing tumor lobes, new tumor blood vessel recruits, the urethra border, and the pelvic node basin. The potential of Flk1-targeted US imaging as a predictive imaging tool was confirmed by correlation studies of three-dimensional US B-mode imaging, gross pathology, and histology analyses. The results of the application in a genetically engineered mouse model with prostate cancer of molecular Flk1-targeted US imaging support the contention that Flk1 can be used as a molecular imaging marker for small tumors undetectable by microimaging and as a molecular diagnostic and prognosis marker for tumor metastasis and progression

Targeting MicroRNA-143 Leads to Inhibition of Glioblastoma Tumor Progression

Glioblastoma (GBM) is the most common and aggressive of all brain tumors, with a median survival of only 14 months after initial diagnosis. Novel therapeutic approaches are an unmet need for GBM treatment. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Several dysregulated miRNAs have been identified in all cancer types including GBM. In this study, we aimed to uncover the role of miR-143 in GBM cell lines, patient samples, and mouse models. Quantitative real-time RT-PCR of RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals. Transient transfection of GBM cells with a miR-143 oligonucleotide inhibitor (miR-143-inh) resulted in reduced cell proliferation, increased apoptosis, and cell cycle arrest. SLC30A8, a glucose metabolism-related protein, was identified as a direct target of miR-143 in GBM cells. Moreover, multiple injections of GBM tumor-bearing mice with a miR-143-inh-liposomal formulation significantly reduced tumor growth compared to control mice. The reduced in vitro cell growth and in vivo tumor growth following miRNA-143 inhibition suggests that miR-143 is a potential therapeutic target for GBM therapy

Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer, highly metastatic, representing 2–4% of all breast cancer cases in the United States. Despite its rare nature, IBC is responsible for 7–10% of all breast cancer deaths, with a 5-year survival rate of 40%. Thus, targeted and effective therapies against IBC are needed. Here, we proposed Lipocalin-2 (LCN2)—a secreted glycoprotein aberrantly abundant in different cancers—as a plausible target for IBC. In immunoblotting, we observed higher LCN2 protein levels in IBC cells than non-IBC cells, where the LCN2 levels were almost undetectable. We assessed the biological effects of targeting LCN2 in IBC cells with small interference RNAs (siRNAs) and small molecule inhibitors. siRNA-mediated LCN2 silencing in IBC cells significantly reduced cell proliferation, viability, migration, and invasion. Furthermore, LCN2 silencing promoted apoptosis and arrested the cell cycle progression in the G0/G1 to S phase transition. We used in silico analysis with a library of 25,000 compounds to identify potential LCN2 inhibitors, and four out of sixteen selected compounds significantly decreased cell proliferation, cell viability, and the AKT phosphorylation levels in SUM149 cells. Moreover, ectopically expressing LCN2 MCF7 cells, treated with two potential LCN2 inhibitors (ZINC00784494 and ZINC00640089) showed a significant decrease in cell proliferation. Our findings suggest LCN2 as a promising target for IBC treatment using siRNA and small molecule inhibitors

Brain Targeted Gold Liposomes Improve RNAi Delivery for Glioblastoma.

INTRODUCTION: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). METHODS: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. RESULTS: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. DISCUSSION: SNA-Liposome-ApoE has the potential to advance the translation of miRNA-based therapies for GBM as well as other CNS disorders