MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX 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 in vitro and in vivo 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 MdmX 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. 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.
- MeSH
- alternativní sestřih účinky léků MeSH
- apoptóza účinky léků MeSH
- down regulace účinky léků MeSH
- enoxacin farmakologie MeSH
- lidé MeSH
- melanom genetika metabolismus patologie MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- nádory prsu genetika metabolismus patologie MeSH
- nádory vaječníků genetika metabolismus patologie MeSH
- ofloxacin farmakologie MeSH
- poškození DNA účinky léků MeSH
- proliferace buněk účinky léků MeSH
- protoonkogenní proteiny c-mdm2 genetika metabolismus MeSH
- signální transdukce účinky léků MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
PURPOSE: Although several molecular markers predicting resistance to cetuximab- or panitumumab-based therapy of metastatic colorectal cancer were described, mutations in RAS proto-oncogenes remain the only predictors being used in daily clinical practice. However, 35%-45% of wild-type RAS patients still do not respond to this anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody-based therapy, and therefore the definition of other predictors forms an important clinical need. The aim of the present retrospective single-institutional study was to evaluate potential genes responsible for resistance to anti-EGFR therapy in relation to mutational analysis of primary versus metastatic lesions. PATIENTS AND METHODS: Twenty-four paired primary and corresponding metastatic tissue samples from eight nonresponding and four responding metastatic colorectal cancer patients treated with cetuximab-based therapy were sequenced using a next-generation sequencing panel of 26 genes involved in EGFR signaling pathway and colorectal carcinogenesis. RESULTS: Mutational status of primary tumors and metastatic lesions was highly concordant in TP53, APC, CTNNB1, KRAS, PIK3CA, PTEN, and FBXW7 genes. Metastatic samples harbor significantly more mutations than primary tumors. Potentially negative predictive value of FBXW7 mutations in relationship to anti-EGFR treatment outcomes was confirmed. Finally, new occurrences of activating KRAS mutations were identified in a group of patients initially determined as wild-type RAS by routinely used qPCR-based RAS mutational tests. All newly detected activating KRAS mutations most likely led to cetuximab treatment failure. CONCLUSION: The results of the present study suggest a need of careful consideration of previously published results of anti-EGFR-targeted therapy with regard to potentially inaccurate diagnostic tools used in the past. Based on our findings, we recommend more extensive use of next-generation sequencing testing in daily clinical practice, as it brings a significant added value in terms of validity of the diagnostic procedure.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: In metastatic colorectal cancer (mCRC), panitumumab is generally considered to be ineffective after the progression on cetuximab therapy. However, few studies have demonstrated that a small subset of mCRC patients may benefit from panitumumab in this setting. PATIENTS AND METHODS: In our study, wild-type KRAS mCRC patients, enrolled into the nationwide Czech registry CORECT between January 2007 and December 2012, were screened for panitumumab therapy after progression on cetuximab. RESULTS: We identified 26 mCRC in the registry with well documented progression on cetuximab in combination with irinotecan-based chemotherapy (FOLFIRI or irinotecan alone) who received panitumumab monotherapy. Partial response (PR) was achieved in 3 (11.5%) patients and stable disease (SD) in 7 (26.9%) patients after 8 weeks of therapy. Thirteen (50.0%) patients had evidence of progressive disease (PD) and in 3 (11.5%) cases response was not available. Furthermore, we confirmed that higher expression levels of newly described biomarker, miR-31-5p, in tumor are significantly associated with shorter progression-free survival (PFS) in patients treated with cetuximab (p=0.038); however, we did not observe association between miR-31-5p and response to panitumumab in mCRC patients after progression on cetuximab. CONCLUSION: It remains possible that a subset of mCRC patients may benefit from panitumumab after progression on cetuximab.
- MeSH
- cetuximab aplikace a dávkování MeSH
- dospělí MeSH
- humanizované monoklonální protilátky MeSH
- kolorektální nádory farmakoterapie genetika patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikro RNA biosyntéza genetika MeSH
- monoklonální protilátky aplikace a dávkování MeSH
- mutace MeSH
- nádorové biomarkery biosyntéza genetika MeSH
- přežití po terapii bez příznaků nemoci MeSH
- prognóza MeSH
- progrese nemoci MeSH
- protoonkogenní proteiny p21(ras) genetika MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
1 svazek : grafy ; 30 cm
In the proposed project we are aiming idetification of the new molecular markers (effector molecules of EGFR signalling, microRNAs) enabling response prediction to anti-EGFR therapy in patients with metastastic colorectal cancer carrying wild-type oncogene KRAS, which can lead not only to better understanding of molecular mechanisms of resistance, but also to individualization of therapy and therefore achievement of better therapeutical results and higher quality of life in patients with metastatic colorectal cancer.
V předkládaném projektu se snažíme nalézt nové molekulární markery (efektorové molekuly signalizace EGFR, mikroRNA) umožňující predikovat odpověď na anti-EGFR terapii ve skupině pacientů s metastatickým kolorektálním karcinomem s nemutovaným onkogenem KRAS, které mohou vést nejen k pochopení dalších molekulárních mechanizmů rezistence, ale také k individualizaci léčby, a tím i k dosažení lepších léčebných výsledků a vyšší kvality života pacientů s metastatickým kolorektálním karcinomem.
- MeSH
- cetuximab MeSH
- cílená molekulární terapie MeSH
- erbB receptory analýza MeSH
- humanizované monoklonální protilátky terapeutické užití MeSH
- individualizovaná medicína MeSH
- kolorektální nádory farmakoterapie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- mikro RNA MeSH
- přežití MeSH
- protoonkogenní proteiny p21(ras) MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- gastroenterologie
- onkologie
- molekulární biologie, molekulární medicína
- farmakoterapie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
- Publikační typ
- abstrakt z konference MeSH
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.
- MeSH
- apoptóza genetika MeSH
- buňky HT-29 MeSH
- Caco-2 buňky MeSH
- cyklin B1 genetika metabolismus MeSH
- dospělí MeSH
- HCT116 buňky MeSH
- Kaplanův-Meierův odhad MeSH
- kolorektální nádory genetika metabolismus patologie MeSH
- kontrolní body fáze G1 buněčného cyklu genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- poly(ADP-ribosa)-polymerasy genetika metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteinkinasa CDC2 genetika metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- RNA dlouhá nekódující genetika metabolismus MeSH
- RNA interference MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- vazba proteinů MeSH
- western blotting MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- antitumorózní látky farmakologie terapeutické užití MeSH
- cetuximab farmakologie terapeutické užití MeSH
- dospělí MeSH
- erbB receptory antagonisté a inhibitory MeSH
- humanizované monoklonální protilátky farmakologie terapeutické užití MeSH
- kolorektální nádory diagnóza farmakoterapie genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- monoklonální protilátky farmakologie terapeutické užití MeSH
- nádorové biomarkery genetika MeSH
- prediktivní hodnota testů MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH