Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer

We determined expression of 83 long non-coding RNAs (lncRNAs) and identified ZFAS1 to be significantly up-regulated in colorectal cancer (CRC) tissue. In cohort of 119 CRC patients we observed that 111 cases displayed at least two-times higher expression of ZFAS1 in CRC compared to paired normal colorectal tissue (P < 0.0001). By use of CRC cell lines (HCT116+/+, HCT116-/- and DLD-1) we showed, that ZFAS1 silencing decreases proliferation through G1-arrest of cell cycle, and also tumorigenicity of CRC cells. We identified Cyclin-dependent kinase 1 (CDK1) as interacting partner of ZFAS1 by pull-down experiment and RNA immunoprecipitation. Further, we have predicted by bioinformatics approach ZFAS1 to sponge miR-590-3p, which was proved to target CDK1. Levels of CDK1 were not affected by ZFAS1 silencing, but cyclin B1 was decreased in both cell lines. We observed significant increase in p53 levels and PARP cleavage in CRC cell lines after ZFAS1 silencing indicating increase in apoptosis. Our data suggest that ZFAS1 may function as oncogene in CRC by two main actions: (i) via destabilization of p53 and through (ii) interaction with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. However, molecular mechanisms behind these interactions have to be further clarified

MicroRNA expression profiling identifies miR-31-5p/3p as associated with time to progression in wild-type RAS metastatic colorectal cancer treated with cetuximab

The aim of our study was to investigate whether microRNAs (miRNAs) could serve as predictive biomarkers to anti-EGFR therapy (cetuximab, panitumumab) in patients with RAS wild-type (wt-RAS) metastatic colorectal cancer (mCRC). Historical cohort of 93 patients with mCRC (2006-2009) was included and further divided into exploratory and validation cohorts. MiRNAs expression profiling was performed on the exploratory cohort of 41 wt-KRAS mCRC patients treated with cetuximab to identify miRNAs associated with time to progression (TTP). The validation was performed on two independent cohorts: 28 patients of wt-RAS mCRC treated with cetuximab and 24 patients of wt-RAS mCRC treated with panitumumab. We identified 9 miRNAs with significantly different expression between responders and non-responders to cetuximab therapy (

Prediction of response to anti-EGFR antibody-based therapies by multigene sequencing in colorectal cancer patients

Background. The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (moAbs) cetuximab or panitumumab are administered to colorectal cancer (CRC) patients who harbor wild-type RAS proto-oncogenes. However, a percentage of patients do not respond to this treatment. In addition to mutations in the RAS genes, mutations in other genes, such as BRAF, PI3KCA, or PTEN, could be involved in the resistance to anti-EGFR moAb therapy. Methods. In order to develop a comprehensive approach for the detection of mutations and to eventually identify other genes responsible for resistance to anti-EGFR moAbs, we investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab. Among these, 37 samples were responsive and 28 were resistant. Results. We confirmed that mutations in EGFR-pathway genes (KRAS, NRAS, BRAF, PI3KCA) were relevant for conferring resistance to therapy and could predict response (p = 0.001). After exclusion of KRAS, NRAS, BRAF and PI3KCA combined mutations could still significantly associate to resistant phenotype (p = 0.045, by Fisher exact test). In addition, mutations in FBXW7 and SMAD4 were prevalent in cases that were non-responsive to anti-EGFR moAb. After we combined the mutations of all genes (excluding KRAS), the ability to predict response to therapy improved significantly (p = 0.002, by Fisher exact test). Conclusions. The combination of mutations at KRAS and at the five gene panel demonstrates the usefulness and feasibility of multigene sequencing to assess response to anti-EGFR moAbs. The application of parallel sequencing technology in clinical practice, in addition to its innate ability to simultaneously examine the genetic status of several cancer genes, proved to be more accurate and sensitive than the presently in use traditional approaches

Enoxacin inhibits melanoma cell growth.

<p>(<b>A</b>) Melanoma cell lines were grown in the presence of enoxacin (50 μg/ml) or 0.2% NaOH (vehicle, negative control). Relative cell growth was determined using standard MTT assay after 1, 2, 3, 4, and 5 days. Results of three independent experiments are presented (means +/- SD). (<b>B</b>) Melanoma cell lines were grown in the presence of 0.2% NaOH (control), enoxacin or ofloxacin (both 50 μg/ml) for four days and relative cell growth was determined by MTT assays. Results of three independent experiments (means + standard deviations) are presented. A significant difference between the effect of enoxacin and ofloxacin **P<0.01 (Student’s t-test, two-tailed). (<b>C</b>) A375 cells were grown for 3 days in the presence of 0.2% NaOH (control) or enoxacin in concentrations 25, 50, 75, and 100 μg/ml. Cell viability was determined using propidium iodide exclusion assay and flow cytometry. Results of three independent experiments are presented (means + SD). ** P<0.01 (Student’s t-test, two-tailed).</p

Enoxacin activates p53-dependent transcription in melanoma cells overexpressing MdmX without inducing DNA damage.

<p>(<b>A, left</b>) A375 cells stably transfected with a p53-responsive luciferase reporter construct were grown for 24 hours in the presence of enoxacin in concentrations 25, 50, 75, and 100 μg/ml. Relative p53 transcriptional activity was determined by measuring the activity of luciferase in cell lysates. Results of three independent experiments are presented (means + SD). * P<0.05, ** P<0.01 (Student’s t-test, two-tailed). (<b>A, right</b>) A375 cells were treated with enoxacin for 24 hours, lysed, and protein levels of MdmX and p53 in cell lysates were analyzed by Western blotting. PCNA served as a loading control. (<b>B</b>) A375 cells stably transfected with a p53 activity reporter construct were treated with 0.2% NaOH (control), etoposide, and three fluoroquinolone antibiotics, and p53 activity was determined. Results of three independent experiments (means + SD) are presented. A significant difference between control and antibiotic-treated cells * P<0.05, ** P<0.01 (Student’s t-test, two-tailed). (<b>C</b>) Quantitative real-time PCR analysis of the expression of selected p53 target genes in A375 cells treated for 24 hours with 50 or 100 μg/ml enoxacin or 0.5 μM doxorubicin. The expression of HPRT1/HGPRT served as an endogenous control. (<b>D</b>) Mel-Juso cells stably transfected with a p53-responsive luciferase reporter construct were grown for 24 hours in the presence of enoxacin in concentrations 50 and 100 μg/ml. Relative p53 activity was determined by measuring luciferase activity in cell lysates. Results of three independent experiments are presented (means + SD). * P<0.05, (Student’s t-test, two-tailed). MdmX and p53 protein levels in Mel-Juso cell lysates were analyzed by Western blotting. PCNA served as a loading control. (<b>E</b>) (<b>F</b>) Western blot analysis of A375 cells treated with increasing concentrations of enoxacin or DNA damaging drugs for the indicated time. A mixture of three different primary monoclonal antibodies recognizing various epitopes in human Mdm2 was used in the Mdm2 panels. * Non-specific band.</p

Enoxacin promotes <i>MdmX</i> exon 6 skipping in melanoma and non-melanoma cancers overexpressing MdmX.

<p>(<b>A</b>) Schematic representation of MdmX mRNA exon structure and binding sites for PCR primers used in RT-PCR to determine the presence/absence of exon 6 (VI). (<b>B, left</b>) Schematic representation of expected <i>MdmX</i> RT-PCR products in the case of presence (<i>MdmX-FL</i>) or absence (<i>MdmX-S</i>) of exon 6. (<b>B, right</b>) RT-PCR analysis of <i>MdmX</i> exon 6 skipping in A375, A2780, and MCF7 cells treated with enoxacin (50 and 100 μg/ml) for 24 hours (primers 1 –top, primers 2 –middle, <i>GAPDH</i> PCR control–bottom). (<b>C</b>) RT-PCR analysis of MdmX exon 6 skipping in A375, A2780 and MCF7 cancer cell lines treated with ciprofloxacin and ofloxacin (both 100 μg/ml) for 24 hours (MdmX primers 2 –top, GAPDH PCR control–bottom). * Non-specific band.</p

Enoxacin induces changes in the expression pattern of a large set of matured miRNAs in A375 cells.

<p>(<b>A</b>) miRNA expression profiling of control and enoxacin-treated A375 cells. Total RNA, including the small RNAs, was analyzed with Affymetrix GeneChip miRNA 3.0 arrays. The heat map shows statistically significant changes in the expression of miRNAs in A375 melanoma cells in response to enoxacin treatment (p<0.05; three independent experiments). (<b>B</b>) Matured miRNAs identified as significantly up- or down-regulated in A375 melanoma cells in response to enoxacin (p < 0.05).</p

Enoxacin promotes MdmX downregulation in different cancer types.

<p>(<b>A</b>) Western blot analysis of changes in cellular MdmX protein levels induced by 24 h treatment with three fluoroquinolone drugs at indicated concentrations in A375 melanoma, A2780 ovarian carcinoma, and MCF7 breast carcinoma. PCNA served as a loading control. (<b>B</b>)(<b>C</b>) Western blot analysis of changes in cellular MdmX, Mdm2, and p53 protein levels induced by 24 h treatment with enoxacin and DNA damaging agents. PCNA served as a loading control. (B) MCF7, (C) A2780, * non-specific band.</p

A small molecule drug promoting miRNA processing induces alternative splicing of <i>MdmX</i> transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein

<div><p>MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of <i>MdmX</i> transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers <i>in vitro</i> and <i>in vivo</i> by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted <i>MdmX</i> exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX.</p><p>Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.</p></div