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

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

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
• 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
• protinádorové látky farmakologie   terapeutické užití   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

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 (P ≤ 0.01). These 9 miRNAs were further evaluated in two independent cohorts of patients and miR-31-3p (P < 0.001) and miR-31-5p (P < 0.001) were successfully confirmed as strongly associated with TTP in wt-RAS mCRC patients treated with cetuximab but not panitumumab. When evaluated on the complete cohort of cetuximab patients (N = 69), miR-31-3p (HR, 5.10; 95% CI, 2.52-10.32; P < 0.001) and miR-31-5p (HR, 4.80; 95% CI, 2.50-9.24; P < 0.001) were correlated with TTP on the comparable level of significance. There was no difference in miR-31-5p/3p expression levels in RAS mutated and wild-type tumor samples. MiR-31-5p/3p are promising predictive biomarkers of cetuximab response in wt-RAS mCRC patients.

• MeSH
• buňky HT-29 MeSH
• cetuximab terapeutické užití   MeSH
• dospělí MeSH
• erbB receptory antagonisté a inhibitory   MeSH
• HCT116 buňky MeSH
• kohortové studie MeSH
• kolorektální nádory farmakoterapie   genetika   metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA biosyntéza   genetika   MeSH
• monoklonální protilátky terapeutické užití   MeSH
• protinádorové látky aplikace a dávkování   terapeutické užití   MeSH
• ras proteiny genetika   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
• práce podpořená grantem MeSH

miRNAs are short noncoding RNAs that post-transcriptionally regulate gene expression. miRNAs' ability to inhibit translation of oncogenes and tumor suppressor genes implies they have an involvement in carcinogenesis. Specific miRNA expression signatures have been identified in a variety of human cancers. More recently, occurrence of miRNAs in the blood serum and plasma of humans has been repeatedly observed. miRNA levels in serum and plasma are more stable, reproducible and consistent among individuals of the same species in comparison with other circulating nucleic acids. Circulating miRNAs have been successfully evaluated in a wide range of solid cancers as promising novel noninvasive biomarkers of early disease onset or relapse. Here we describe the origin of circulating miRNAs, principles of their immense stability and proposed functions, and comprehensively summarize studies focusing on their significance in the most frequently studied cancer types in this regard, including breast, colorectal, lung and prostate cancer.

• MeSH
• kolorektální nádory krev   MeSH
• lidé MeSH
• mikro RNA krev   MeSH
• nádorové biomarkery krev   MeSH
• nádory plic krev   MeSH
• nádory prostaty krev   MeSH
• nádory prsu krev   MeSH
• stabilita RNA MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

MicroRNAs (miRNAs) are short, 18-25-nucleotide long, non-coding single-stranded RNAs, which are capable to regulate gene expression on post-transcriptional level through binding to their target protein-encoding mRNAs. miRNAs regulate individual components of multiple oncogenic pathways. One of them is epidermal growth factor receptor (EGFR) signalling pathway that regulates cell proliferation, differentiation, migration, angiogenesis and apoptosis. All these processes are deregulated in colorectal cancer (CRC). Moreover, EGFR has been validated as the therapeutic target in CRC, and monoclonal antibodies cetuximab and panitumumab are used in the therapy of patients with metastatic CRC. Because of the extensive involvement of miRNAs in the regulation of EGFR signalling, it seems they could also serve as promising predictive biomarkers to anti-EGFR therapy. In this review, we summarize current knowledge about miRNAs targeting EGFR signalling pathway, their functioning in CRC pathogenesis and potential usage as biomarkers.

• MeSH
• erbB receptory genetika   metabolismus   MeSH
• kolorektální nádory genetika   metabolismus   MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• nádorové biomarkery genetika   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• RNA interference MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Většinu eukaryotického genomu představují DNA sekvence, které nekódují proteiny. Tyto sekvence jsou přepisovány buď podle vývojového programu daného organizmu nebo v rámci odpovědi na vnější signály. Výsledkem transkripce takových sekvencí je pak velké množství dlouhých nekódujících RNA (lncRNA). Celogenomové studie předpokládají existenci více než 3 300 lncRNA. Dlouhé nekódující RNA jsou definovány jako molekuly nekódujících RNA o délce více než 200 nukleotidů. Vzhledem k vysoké míře komplexnosti a rozmanitosti těchto sekvencí byl nárůst poznání v této oblasti relativně pomalý. Ačkoli bylo dosud funkčně charakterizováno pouze omezené množství lncRNA, jejich regulační potenciál je již dnes evidentní. LncRNA hrají klíčové role jak v transkripčních, tak v post-transkripčních regulačních drahách. U mnoha nádorových onemocnění dochází k deregulaci lncRNA, což společně s jejich funkčními vlastnostmi naznačuje jejich významný potenciál v procesech maligní transformace. Tento přehledový článek je zaměřen na shrnutí nedávno objevených skupin lncRNA, popis jejich biologických funkcí a zejména na jejich význam v nádorové biologii a translačním onkologickém výzkumu.

A major portion of the eukaryotic genome is occupied by DNA sequences; transcripts of these sequences do not code for proteins. This part of the eukaryotic genome is transcribed in a developmentally regulated manner or as a response to external stimuli to produce large numbers of long non-coding RNAs (lncRNAs). Genome-wide studies indicate the existence of more than 3,300 lncRNAs. Long non-coding RNAs are tentatively defined as molecules of ncRNAs that are more than two hundred nucleotides long. Due to the complexity and diversity of their sequences, progress in the field of lncRNAs has been very slow. Nonetheless, lncRNAs have emerged as key molecules involved in the control of transcriptional and posttranscriptional gene regulatory pathways. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of lncRNAs is already evident, emphasizing that a genome-wide characterization of functional lncRNAs is needed. The fact that many lncRNAs are deregulated in various human cancers, together with their functional characteristics, implies their eminent role in carcinogenesis. In this review, we summarize novel classes of lncRNAs, describe their biological functions emphasizing their roles in tumor biology and translational oncology research.

• Klíčová slova
• lincRNA, T-UC,
• MeSH
• 3' nepřekládaná oblast fyziologie   genetika   imunologie   MeSH
• 5' nepřekládaná oblast fyziologie   genetika   imunologie   MeSH
• financování organizované MeSH
• genetické markery genetika   MeSH
• genetické struktury MeSH
• genom lidský fyziologie   genetika   imunologie   MeSH
• hepatocelulární karcinom diagnóza   genetika   MeSH
• lidé MeSH
• malá nekódující RNA genetika   izolace a purifikace   MeSH
• mikro RNA genetika   izolace a purifikace   MeSH
• nádory prostaty diagnóza   genetika   MeSH
• nádory prsu diagnóza   genetika   MeSH
• nádory diagnóza   etiologie   genetika   MeSH
• nekódující RNA diagnostické užití   genetika   izolace a purifikace   MeSH
• nepřekládané oblasti fyziologie   genetika   imunologie   MeSH
• proteiny vázající telomery genetika   MeSH
• pseudogeny fyziologie   genetika   imunologie   MeSH
• translační biomedicínský výzkum metody   trendy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

BACKGROUND: Rectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy with fluoropyrimidines (capecitabine or 5-fluorouracil) followed by radical surgical resection. Unfortunately, a significant proportion of tumors do not respond enough to the neoadjuvant treatment and these patients are at risk of relapse. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers for rectal cancer patients. METHODS: We selected 20 patients who underwent neoadjuvant chemoradiotherapy for advanced rectal cancer and whose tumors were classified as most sensitive or resistant to the treatment. These two groups were compared using large-scale miRNA expression profiling. RESULTS: Expression levels of 8 miRNAs significantly differed between two groups. MiR-215, miR-190b and miR-29b-2* have been overexpressed in non-responders, and let-7e, miR-196b, miR-450a, miR-450b-5p and miR-99a* have shown higher expression levels in responders. Using these miRNAs 9 of 10 responders and 9 of 10 non-responders (p < 0.05) have been correctly classified. CONCLUSIONS: Our pilot study suggests that miRNAs are part of the mechanisms that are involved in response of rectal cancer to the chemoradiotherapy and that miRNAs may be promising predictive biomarkers for such patients. In most miRNAs we identified (miR-215, miR-99a*, miR-196b, miR-450b-5p and let-7e), the connection between their expression and radioresistance or chemoresistance to inhibitors of thymidylate synthetase was already established.

• MeSH
• adenokarcinom genetika   metabolismus   patologie   chirurgie   terapie   MeSH
• chemoradioterapie * MeSH
• chemorezistence genetika   MeSH
• deoxycytidin analogy a deriváty   farmakologie   terapeutické užití   MeSH
• dospělí MeSH
• fluoruracil analogy a deriváty   farmakologie   terapeutické užití   MeSH
• kombinovaná terapie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA biosyntéza   genetika   MeSH
• nádorové proteiny antagonisté a inhibitory   fyziologie   MeSH
• nádory rekta genetika   metabolismus   patologie   chirurgie   terapie   MeSH
• neoadjuvantní terapie * MeSH
• pilotní projekty MeSH
• protinádorové antimetabolity farmakologie   terapeutické užití   MeSH
• regulace genové exprese u nádorů * MeSH
• retrospektivní studie MeSH
• RNA nádorová biosyntéza   genetika   MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• thymidylátsynthasa antagonisté a inhibitory   fyziologie   MeSH
• tolerance záření genetika   MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH