MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to ...have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gene silencing by DNA hypermethylation of CpG islands is a well‐characterized phenomenon in cancer. The effect of hypomethylation in particular of non‐CpG island genes is much less well described. By ...genome‐wide screening, we identified 105 genes in microsatellite stable (MSS) colorectal adenocarcinomas with an inverse correlation (Spearman's ρ ≤ −0.40) between methylation and expression. Of these, 35 (33%) were hypomethylated non‐CpG island genes and two of them, APOLD1 (Spearman's ρ = −0.82) and SRPX2 (Spearman's ρ = −0.80) were selected for further analyses. Hypomethylation of both genes were localized events not shared by adjacent genes. A set of 662 FFPE DNA samples not only confirmed that APOLD1 and SRPX2 are hypomethylated in CRC but also revealed hypomethylation to be significantly (p < 0.01) associated with tumors being localized in the left side, CpG island methylator phenotype negative, MSS, BRAF wt, undifferentiated and of adenocarcinoma histosubtype. Demethylation experiments supported SRPX2 being epigenetically regulated via DNA methylation, whereas other mechanisms in addition to DNA methylation seem to be involved in the regulation of APOLD1. We further identified miR‐149 as a potential novel post‐transcriptional regulator of SRPX2. In carcinoma tissue, miR‐149 was downregulated and inversely correlated to SRPX2 (ρ = −0.77). Furthermore, ectopic expression of miR‐149 significantly reduced SRPX2 transcript levels. Our study highlights that in colorectal tumors, hypomethylation of non‐CpG island‐associated promoters deregulate gene expression nearly as frequent as do CpG‐island hypermethylation. The hypomethylation of SRPX2 is focal and not part of a large block. Furthermore, it often translates to an increased expression level, which may be modulated by miR‐149.
What's new?
DNA hypo‐ and hypermethylation both occur frequently in colorectal cancer, yet so far most attention has focused on the role of hypermethylation in silencing critical genes with CpG island promoters. This study provides evidence that hypomethylation of non‐CpG island promoters deregulate gene expression nearly as frequently as does the hypermethylation of CpG island promoters. The authors identified 35 genes whose non‐CpG island promoters were hypomethylated and expression levels inversely correlated. Hypomethylation of SRPX2 also correlated with poorly differentiated tumors, indicating its important role during CRC progression. The authors further identified miR‐149 as a potential novel post‐transcriptional regulator of SRPX2.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR‐200 family (miR‐200a, ‐200b, ‐200c, ‐141 and ‐429) and miR‐205 are frequently silenced in advanced cancer and have ...been implicated in epithelial to mesenchymal transition (EMT) and tumor invasion by targeting the transcriptional repressors of E‐cadherin, ZEB1 and ZEB2. ZEB1 is also known to repress miR‐200c‐141 transcription in a negative feedback loop, but otherwise little is known about the transcriptional regulation of the miR‐200 family and miR‐205. Recently, miR‐200 silencing was also reported in cancer stem cells, implying that miR‐200 deregulation is a key event in multiple levels of tumor biology. However, what prevents miR‐200 expression remains largely unanswered. Here we report concerted transcriptional regulation of the miR‐200 and miR‐205 loci in bladder tumors and bladder cell lines. Using a combination of miRNA expression arrays, qPCR assays and mass spectrometry DNA methylation analyses, we show that the miR‐200 and miR‐205 loci are specifically silenced and gain promoter hypermethylation and repressive chromatin marks in muscle invasive bladder tumors and undifferentiated bladder cell lines. Moreover, we report that miR‐200c expression is significantly correlated with early stage T1 bladder tumor progression, and propose miR‐200 and miR‐205 silencing and DNA hypermethylation as possible prognostic markers in bladder cancer. In addition, we observe that the mesoderm transcription factor TWIST1 and miR‐200 expression are inversely correlated in bladder tumor samples and cell lines. TWIST1 associates directly with the miR‐200 and miR‐205 promoters, and may act as a repressor of miR‐200 and miR‐205 expression.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Epigenetic alterations are common and can now be addressed in a parallel fashion. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in ...metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers.
A microarray assay was utilized to analyze methylation in 56 samples. Independent validation was conducted in 63 samples by a PCR-based method and bisulfite sequencing. The methylation levels in 174 urine specimens were quantified. Transcript levels were analyzed using expression microarrays and pathways were analyzed using dedicated software.
Global methylation patterns were established within and outside CpG islands. We validated methylation of the eight tumor markers genes ZNF154 (P < 0.0001), HOXA9 (P < 0.0001), POU4F2 (P < 0.0001), EOMES (P = 0.0005), ACOT11 (P = 0.0001), PCDHGA12 (P = 0.0001), CA3 (P = 0.0002), and PTGDR (P = 0.0110), the candidate marker of disease progression TBX4 (P < 0.04), and other genes with stage-specific methylation. The methylation of metachronous tumors was stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (P < 0.0001) and specifically hypomethylation of keratins, which together with keratin-like proteins were epigenetically regulated. In DNA from voided urine, we detected differential methylation of ZNF154 (P < 0.0001), POU4F2 (P < 0.0001), HOXA9 (P < 0.0001), and EOMES (P < 0.0001), achieving 84% sensitivity and 96% specificity.
We initiated a detailed mapping of the methylome in metachronous bladder cancer. Novel genes with tumor, chromosome, as well as pathway-specific differential methylation in bladder cancer were identified. The methylated genes were promising cancer markers for early detection of bladder cancer.
Abstract
Background. Adjuvant chemotherapy is established routine therapy for colon cancer (CC) patients with radically resected stage III and 'high-risk' stage II disease. The decision on ...recommending adjuvant chemotherapy, however, is based on data from older patient cohorts not reflecting improvements in pre-operative staging, surgery, and pathological examination. The aim is to review the current risk of recurrence in stage II and III patients and second, to estimate the relative importance of routinely assessed clinico-pathological variables.
Methods. The PubMed/MEDLINE and the Cochrane databases were systematically searched for randomized controlled studies and observational studies published after 1 January 2005 with patients included after January 1995 on prognosis in surgically treated stage II and III CC patients.
Results. Of 2596 studies identified, 37 met the inclusion criteria and 25 provided data for meta-analysis. The total patient sample size in the 25 studies reporting either disease-free (DFS) or recurrence-free survival was 15 559 in stage II and 18 425 in stage III. Five-year DFS for stage II patients operated without subsequent adjuvant chemotherapy was 81.4% 95% confidence interval (CI) 75.4-87.4; in studies with good/very good quality of reporting 82.7%, (95% CI 80.8-84.6). For stage II patients treated with adjuvant chemotherapy, the five-year DFS was 79.3% (95% CI 75.6-83.1). For stage III patients without chemotherapy, five-year DFS was 49.0% (95% CI 23.2-74.8) and for those treated with adjuvant chemotherapy, 63.6% (95% CI 59.3-67.9). The prognostic impact of commonly investigated clinico-pathological parameters, (pT-stage, pN-stage, differentiation, number of lymph nodes studied, MMR-status, and emergency surgery) were confirmed.
Conclusions. In this meta-analysis, studies with good quality of reporting show a five-year DFS of 82.7% for stage II CC without adjuvant chemotherapy, whereas the five-year DFS is 63.8% for stage III CC with adjuvant chemotherapy. Due to insufficient reporting on treatment quality the presented DFS is likely an under-estimation of what is achieved at high-quality centers today.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In our study, whole‐genome methylation arrays were applied to identify novel genes with tumor specific DNA methylation of promoter CpG islands in pre‐malignant and malignant colorectal lesions. Using ...a combination of Illumina HumanMethylation27 beadchips, Methylation‐Sensitive High Resolution Melting (MS‐HRM) analysis, and Exon arrays (Affymetrix) the DNA methylation pattern of ∼14,000 genes and their transcript levels were investigated in six normal mucosas, six adenomas and 30 MSI and MSS carcinomas. Sixty eight genes with tumor‐specific hypermethylation were identified (p < 0.005). Identified hypermethylated sites were validated in an independent sample set of eight normal mucosas, 12 adenomas, 40 MSS and nine MSI cancer samples. The methylation patterns of 15 selected genes, hypermethylated in adenomas and carcinomas (FLI1, ST6GALNAC5, TWIST1, ADHFE1, JAM2, IRF4, CNRIP1, NRG1 and EYA4), in carcinomas only (ABHD9, AOX1 and RERG), or in MSI but not MSS carcinomas (RAMP2, DSC3 and MLH1) were validated using MS‐HRM. Four of these genes (MLH1, AOX1, EYA4 and TWIST1) had previously been reported to be hypermethylated in CRC. Eleven genes, not previously known to be affected by CRC specific hypermethylation, were identified and validated. Inverse correlation to gene expression was observed for six of the 15 genes with Spearman correlation coefficients ranging from −0.39 to −0.60. For six of these genes the altered methylation patterns had a profound transcriptional association, indicating that methylation of these genes may play a direct regulatory role. The hypermethylation changes often occurred already in adenomas, indicating that they may be used as biomarkers for early detection of CRC.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The backbone of current cytotoxic treatment of metastatic colorectal cancer (mCRC) consists of a fluoropyrimidine together with either oxaliplatin (XELOX/FOLFOX) or irinotecan (XELIRI/FOLFIRI). With ...an overall objective response rate of approximately 50% for either treatment combination, a major unsolved problem is that no predictors of response to these treatments are available. To address this issue, we profiled 742 microRNAs in laser-capture microdissected cancer cells from responding and non-responding patients receiving XELOX/FOLFOX as first-line treatment for mCRC, and identified, among others, high expression of miR-625-3p, miR-181b and miR-27b to be associated with poor clinical response. In a validation cohort of 94 mCRC patients treated first-line with XELOX, high expression of miR-625-3p was confirmed to be associated with poor response (OR = 6.25, 95%CI 1.8; 21.0). Independent analyses showed that miR-625-3p was not dysregulated between normal and cancer samples, nor was its expression associated with recurrence of stage II or III disease, indicating that miR-625-3p solely is a response marker. Finally, we also found that these miRNAs were up-regulated in oxaliplatin resistant HCT116/oxPt (miR-625-3p, miR-181b and miR-27b) and LoVo/oxPt (miR-181b) colon cancer cell lines as compared with their isogenic parental cells. Altogether, our results suggest an association between miR-625-3p and response to first-line oxaliplatin based chemotherapy of mCRC.
► High miR-625-3p levels were associated with resistance to oxaliplatin based chemotherapy and a shortened overall survival. ► High miR-625-3p levels were not prognostic, but solely predictive of oxaliplatin response. ► miR-625-3p was up-regulated in an oxaliplatin resistant colon cancer cell line.
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FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background: Robust optimization has been suggested as an approach to reduce the irradiated volume in lung Stereotactic Body Radiation Therapy (SBRT). We performed a retrospective planning study to ...investigate the potential benefits over Planning Target Volume (PTV)-based planning. Material and methods: Thirty-nine patients had additional plans using robust optimization with 5-mm isocenter shifts of the Gross Tumor Volume (GTV) created in addition to the PTV-based plan used for treatment. The optimization included the mid-position phase and the extreme breathing phases of the 4D-CT planning scan. The plans were compared for tumor coverage, isodose volumes, and doses to Organs At Risk (OAR). Additionally, we evaluated both plans with respect to observed tumor motion using the peak tumor motion seen on the planning scan and cone-beam CTs. Results: Statistically significant reductions in irradiated isodose volumes and doses to OAR were achieved with robust optimization, while preserving tumor dose. The reductions were largest for the low-dose volumes and reductions up to 188 ccm was observed. The robust evaluation based on observed peak tumor motion showed comparable target doses between the two planning methods. Accumulated mean GTV-dose was increased by a median of 4.46 Gy and a non-significant increase of 100 Monitor Units (MU) was seen in the robust optimized plans. Interpretation: The robust plans required more time to prepare, and while it might not be a feasible planning strategy for all lung SBRT patients, we suggest it might be useful for selected patients.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Approximately half of all human genes use alternative transcription start sites (TSSs) to control mRNA levels and broaden the transcriptional output in healthy tissues. Aberrant expression patterns ...promoting carcinogenesis, however, may arise from alternative promoter usage.
By profiling 108 colorectal samples using exon arrays, we identified nine genes (TCF12, OSBPL1A, TRAK1, ANK3, CHEK1, UGP2, LMO7, ACSL5, and SCIN) showing tumor-specific alternative TSS usage in both adenoma and cancer samples relative to normal mucosa. Analysis of independent exon array data sets corroborated these findings. Additionally, we confirmed the observed patterns for selected mRNAs using quantitative real-time reverse-transcription PCR. Interestingly, for some of the genes, the tumor-specific TSS usage was not restricted to colorectal cancer. A comprehensive survey of the nine genes in lung, bladder, liver, prostate, gastric, and brain cancer revealed significantly altered mRNA isoform ratios for CHEK1, OSBPL1A, and TCF12 in a subset of these cancer types.To identify the mechanism responsible for the shift in alternative TSS usage, we antagonized the Wnt-signaling pathway in DLD1 and Ls174T colorectal cancer cell lines, which remarkably led to a shift in the preferred TSS for both OSBPL1A and TRAK1. This indicated a regulatory role of the Wnt pathway in selecting TSS, possibly also involving TP53 and SOX9, as their transcription binding sites were enriched in the promoters of the tumor preferred isoforms together with their mRNA levels being increased in tumor samples. Finally, to evaluate the prognostic impact of the altered TSS usage, immunohistochemistry was used to show deregulation of the total protein levels of both TCF12 and OSBPL1A, corresponding to the mRNA levels observed. Furthermore, the level of nuclear TCF12 had a significant correlation to progression free survival in a cohort of 248 stage II colorectal cancer samples.
Alternative TSS usage in colorectal adenoma and cancer samples has been shown for nine genes, and OSBPL1A and TRAK1 were found to be regulated in vitro by Wnt signaling. TCF12 protein expression was upregulated in cancer samples and correlated with progression free survival.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
Circulating DNA can be detected and quantified in the blood of cancer patients and used for detection of tumor‐specific genetic alterations. The clinical utility has been intensively ...investigated for the past 10 years. The majority of reports focus on analyzing the clinical potential of tumor‐specific mutations, whereas the use of total cell‐free DNA (cfDNA) quantification is somehow controversial and sparsely described in the literature, but holds important clinical information in itself. The purpose of the present report was to present a systematic review and meta‐analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection.
Materials and Methods
A systematic literature search of PubMed and Embase was performed by two independent investigators. Eligibility criteria were (a) total cfDNA analysis, (b) mCRC, and (c) prognostic value during palliative treatment. The preferred reporting items for systematic reviews and meta‐analyses (PRISMA) guidelines were followed, and meta‐analysis applied on both aggregate data extraction and individual patients’ data.
Results
Ten eligible cohorts were identified, including a total of 1,076 patients. Seven studies used quantitative polymerase chain reaction methods, two BEAMing beads, emulsification, amplification, and magnetics technology, and one study digital droplet polymerase chain reaction. The baseline levels of cfDNA was similar in the presented studies, and all studies reported a clear prognostic value in favor of patients with lowest levels of baseline cfDNA. A meta‐analysis revealed a combined estimate of favorable overall survival hazard ratio (HR) in patients with levels below the median cfDNA (HR = 2.39, 95% confidence interval 2.03–2.82, p < .0001).
Conclusion
The total cfDNA levels are high in patients with mCRC and bear strong prognostic information, which should be tested prospectively by using a predefined cut‐off value based on normal values in healthy cohorts. Finally, the potential use of cfDNA for detection of tumor‐specific mutations was emphasized in a large individual patients’ data meta‐analysis.
Implications for Practice
Reliable prognostic markers could help to guide patients and treating physicians regarding the relevance and choice of systemic therapy. Small fragments of circulating cell‐free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta‐analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients with the lowest pre‐treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell‐free DNA analysis can also be used for detection of tumor‐specific mutations, and hold potential as a valuable tool in colorectal cancer treatment.
Recently refined methods for cfDNA quantification and detection of mutations have been developed. Results of clinical investigations of the predictive and prognostic value of total cfDNA and plasma mutations in mCRC suggest a strong prognostic value of baseline plasma levels of cfDNA in this setting. This article presents a systematic review and meta‐analysis of the prognostic value of total plasma cfDNA in patients with mCRC treated with chemotherapy and/or targeted agents.
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