Molecular Classification of Colorectal Cancers and Clinical Application

The molecular genetics of colorectal cancers (CRCs) is among the best understood of common human cancers. It is difficult to predict the prognosis and/or to predict chemoresponding in CRC patients. At present, prognosis is based predominantly on the tumor stage and pathological examination of the disease. Molecular classification of CRCs, based on genomics and transcriptomics, proposed that CRCs can be classified into at least three-to-six subtypes, depending on the gene expression pattern, and groups of marker genes representing to each subtype have also been reported. Gene expression-based subtyping is now widely accepted as a relevant source of disease stratification. We reviewed the previous studies on CRC subtyping, international consortium dedicated to large-scale data sharing and analytics recently established four consensus molecular subtypes with distinguishing features. Predictive markers identified in these studies are under investigation and large-scale clinical evaluations of molecular markers are currently in progress.ope

mRNAs containing NMD-competent premature termination codons are stabilized and translated under UPF1 depletion

mRNAs containing premature termination codons (PTCs) are rapidly degraded through nonsense-mediated mRNA decay (NMD). However, some PTC-containing mRNAs evade NMD, and might generate mutant proteins responsible for various diseases, including cancers. Using PTC-containing human genomic β-globin constructs, we show that a fraction (~30%) of PTC-containing mRNAs expressed from NMD-competent PTC-containing constructs were as stable as their PTC-free counterparts in a steady state. These PTC-containing mRNAs were monosome-enriched and rarely contributed to expression of mutant proteins. Expression of trace amounts of mutant proteins from NMD-competent PTC-containing constructs was not affected by inhibition of eIF4E-dependent translation and such expression was dependent on a continuous influx of newly synthesized PTC-containing mRNAs, indicating that truncated mutant proteins originated primarily in the pioneer round of translation. The generation of mutant proteins was promoted by UPF1 depletion, which induced polysome association of PTC-containing mRNAs, increased eIF4E-bound PTC-containing mRNA levels, and subsequent eIF4E-dependent translation. Our findings suggest that PTC-containing mRNAs are potent and regulatable sources of mutant protein generation.ope

Selective translational regulation of premature termination codon containing mutant mRNAs

의과대학/박사Abnormal mRNAs containing premature termination codons (PTCs) are normally degraded through nonsense-mediated mRNA decay (NMD) at the posttranscriptional level, thus preventing the production of potentially harmful truncated mutant proteins. However, NMD is known to be imperfect. The mRNAs containing PTCs in the last exon (NMD-irrelevant) are not recognized by NMD, so they are not degraded. About 10%-30% of NMD-competent PTC-containing mRNAs are resistant to NMD (NMD-resistant) and exist as stably as their PTC-free counterparts. Moreover, if NMD is inhibited by endogenous or exogenous ways, PTC-containing mRNAs to be degraded are expected to be rescued from NMD (rescued PTC-containing mRNAs). These NMD-irrelevant PTC-containing mRNAs, NMD-resistant PTC-containing mRNAs and rescued PTC-containing mRNAs from NMD are the potential sources for the generation of mutant proteins. Therefore, it has been urged to clarify whether mutant proteins are generated from the PTC-containing mRNAs, but detailed molecular mechanisms have been poorly understood. In this study, to clarify whether mutant proteins from the NMD-irrelevant PTC-containing mRNAs are produced, I identified 29 genes with frequent frameshift mutations in the last exon in colon cancers with high microsatellite instability (MSI-H) and selected three genes (TTK, TCF7L2, and MARCKS) for NMD-irrelevant PTC-containing mRNAs and protein expression analysis. The NMD-irrelevant PTC-containing mRNAs from these mutated genes were not degraded by NMD. However, only faint amounts of endogenous mutant TTK and TCF7L2 were detected, and I failed to detect endogenous mutant MARCKS. By polysome analysis, I demonstrated that NMD-irrelevant PTC-containing mRNAs are actively translated and inhibition of the proteasomal degradation facilitated the rescue of endogenous mutant TTK, TCF7L2, and MARCKS. By comparing the expression of neopeptide-containing or neopeptide-lacking truncated mutant proteins derived from genomic MARCKS constructs containing nonsense or frameshift mutations, I demonstrated that the enhanced degradation of these mutant proteins was driven by neopeptides. To demonstrate the generation of mutant proteins from NMD-resistant PTC-containing mRNAs and rescued PTC-containing mRNAs from NMD, human genomic β-globin expression constructs with nonsense mutations were used in order to avoid enhanced degradation of neopeptide-containing mutant proteins derived from frameshift mutations and I found that about 30 % of PTC-containing mRNAs are resistant to NMD in steady-state and these NMD-resistant PTC-containing mRNAs showed similar stability with their PTC-free counterparts. I demonstrated that the NMD-resistant PTC-containing mRNAs were translationally repressed and therefore, only trace amount of detected mutant proteins was generated from the pioneer round of translation step. Moreover, I found that PTC-containing mRNAs were significantly rescued when NMD was inhibited by down-regulating hUPF1, a key NMD factor and a large amount of mutant proteins from the rescued PTC-containing mRNAs was generated from the bulky translation step. I further demonstrated that down-regulation of UPF1 plays key roles to relieve the translational repression of PTC-containing mRNAs. In conclusion, I found 1) NMD-irrelevant mutant mRNAs containing PTC in the last exon are not degraded by NMD in cells and translated efficiently. 2) Truncated proteins containing neopeptides are rarely detected because of extensively degradation by the ubiquitin？proteasome system which is caused by neopeptides. 3) About 30% of PTC-containing mRNAs from NMD-competent β-globin expression constructs are resistant to NMD and exist as stably as their PTC free counterparts. 4) NMD-resistant PTC-containing mRNAs are translationally repressed and trace amount of mutant proteins from them is mainly generated from the pioneer round of translation step. 5) Mutant proteins from the rescued PTC-containing mRNAs from NMD by UPF1 down-regulation are mostly generated from bulky translation. 6) UPF1 plays key roles in the selective translational regulation of PTC-containing mRNAs depending on NMD status. These findings suggest that PTC-containing mRNAs are the pool of the sources for the mutant protein generation and are subject to differential translational and posttranslational regulations depending on NMD status.ope

Genomic Grade Index predicts postoperative clinical outcome of GIST

Background : Prognosis of localised gastrointestinal stromal tumour (GIST) is heterogeneous, notably for patients with AFIP intermediate or high risk of relapse, who are candidates to adjuvant imatinib. We hypothesised that gene expression profiles might improve the prognostication and help to refine the indications for imatinib. Methods : We collected gene expression and histoclinical data of 146 pre-treatment localised GIST samples treated with surgery alone. We searched for a gene expression signature (GES) predictive for relapse-free survival (RFS) and compared its performances to that of three published prognostic proliferation-based GES (Genomic Grade Index (GGI), 16-Kinase, and CINSARC) and AFIP classification. We also analysed a data set from 28 patients with advanced GIST treated with neo-adjuvant imatinib. Results : We identified a 275-gene GES (gene expression signature) predictive of RFS in a learning set and validated its robustness in an independent set. However, the GGI outperformed its prognostic performances, and those of the two other signatures and the AFIP intermediate-risk classification in two independent tests sets in uni- and multivariate analyses. Importantly, GGI could split the AFIP intermediate/high-risk samples into two groups with different RFS. Genomic Grade Index 'high-risk' tumours were more proliferative and genetically unstable than 'low-risk' tumours, and more sensitive to imatinib. Conclusion : GGI refines the prediction of RFS in localised GIST and might help tailor adjuvant imatinib.ope

MicroRNA-494 downregulates KIT and inhibits gastrointestinal stromal tumor cell proliferation

PURPOSE: Gain-of-function mutations and KIT overexpression are well-known tumorigenesis mechanisms in gastrointestinal stromal tumors (GIST). This study aimed to discover microRNAs (miRNA) that target KIT and reveal the relationship between the discovered miRNAs and KIT expression in GISTs. EXPERIMENTAL DESIGN: Fresh-frozen GISTs from 31 patients were used to confirm the relationship between miR-494 and KIT expression using quantitative reverse transcription-PCR to assess miR-494 expression levels and Western blotting to assess KIT protein expression levels. A luciferase assay was conducted for the target evaluation. The functional effects of miR-494 on GIST882 cells (GIST cell line with activating KIT mutation) were validated by a cell proliferation assay and fluoresce-activated cell sorting analysis. RESULTS: An inverse relationship was found between the expression levels of miR-494 and KIT in GISTs (r = -0.490, P = 0.005). The direct targeting of KIT by miR-494 was shown by the reduction in KIT expression after miR-494 overexpression and the increase in KIT expression after inhibiting endogenous miR-494 expression. We showed that miR-494 regulates KIT by binding two different seed match sites. Induced miR-494 overexpression in GIST882 reduced the expression of downstream molecules in KIT signaling transduction pathways, including phospho-AKT and phospho-STAT3. Finally, miR-494 overexpression provoked apoptosis and inhibited GIST cell growth, which were accompanied by changes in G(1) and S phase content. CONCLUSION: Our findings indicate that miR-494 is a negative regulator of KIT in GISTs and overexpressing miR-494 in GISTs may be a promising approach to GIST treatment.ope

Survivin is a novel transcription regulator of KIT and is downregulated by miRNA-494 in gastrointestinal stromal tumors

Gain-of-function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT-independent targets of KIT-regulating mRNAs. We sought to investigate how miR-494 inhibits GIST proliferation and to identify novel target gene. We used microarray-based gene expression analyses to identify pathways and target genes affected by miR-494. The expressional relationship between survivin and miR-494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR-494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR-494, and its expression showed an inverse correlation with miR-494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = -0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR-494-mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR-494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT.ope

Promoter methylation of PCDH10 by HOTAIR regulates the progression of gastrointestinal stromal tumors

HOTAIR, a long non-coding RNA (lncRNA), plays a crucial role in tumor initiation and metastasis by interacting with the PRC2 complex and the modulation of its target genes. The role of HOTAIR in gastrointestinal stromal tumors (GISTs) is remains unclear. Herein we investigate the mechanism of HOTAIR in the genesis and promotion of GISTs. The expression of HOTAIR was found to be higher in surgically resected high-risk GISTs than that in low- and intermediate-risk GISTs. Using GIST-T1 and GIST882 cells, we demonstrated that HOTAIR repressed apoptosis, was associated with cell cycle progression, and controlled the invasion and migration of GIST cells. Using a gene expression microarray and lists of HOTAIR-associated candidate genes, we suggested that protocadherin 10 (PCDH10) is a key molecule. PCDH10 expression was significantly decreased in GIST-T1 and GIST882 cells, possibly as a consequence of hypermethylation. We observed that HOTAIR induced PCDH10 methylation in a SUZ12-dependent manner. In this study, we found that the malignant character of GISTs was initiated and amplified by PCDH10 in a process regulated by HOTAIR. In summary, our findings imply that PCDH10 and HOTAIR may be useful markers of disease progression and therapeutic targets.ope

Identification of specifically activated angiogenic molecules in HMGB-1-induced angiogenesis

High-mobility group box-1 (HMGB-1) is expressed in almost all cells, and its dysregulated expression correlates with inflammatory diseases, ischemia, and cancer. Some of these conditions accompany HMGB-1-mediated abnormal angiogenesis. Thus far, the mechanism of HMGB-1-induced angiogenesis remains largely unknown. In this study, we performed time-dependent DNA microarray analysis of endothelial cells (ECs) after HMGB-1 or VEGF treatment. The pathway analysis of each gene set upregulated by HMGB-1 or VEGF showed that most HMGB-1-induced angiogenic pathways were also activated by VEGF, although the activation time and gene sets belonging to the pathways differed. In addition, HMGB-1 upregulated some VEGFR signaling-related angiogenic factors including EGR1 and, importantly, novel angiogenic factors, such as ABL2, CEACAM1, KIT, and VIPR1, which are reported to independently promote angiogenesis under physiological and pathological conditions. Our findings suggest that HMGB-1 independently induces angiogenesis by activating HMGB-1-specific angiogenic factors and also functions as an accelerator for VEGF-mediated conventional angiogenesis.ope

β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

WNT signaling activation in colorectal cancers (CRCs) occurs through APC inactivation or β-catenin mutations. Both processes promote β-catenin nuclear accumulation, which up-regulates epithelial-to-mesenchymal transition (EMT). We investigated β-catenin localization, transcriptome, and phenotypic differences of HCT116 cells containing a wild-type (HCT116-WT) or mutant β-catenin allele (HCT116-MT), or parental cells with both WT and mutant alleles (HCT116-P). We then analyzed β-catenin expression and associated phenotypes in CRC tissues. Wild-type β-catenin showed membranous localization, whereas mutant showed nuclear localization; both nuclear and non-nuclear localization were observed in HCT116-P. Microarray analysis revealed down-regulation of Claudin-7 and E-cadherin in HCT116-MT vs. HCT116-WT. Claudin-7 was also down-regulated in HCT116-P vs. HCT116-WT without E-cadherin dysregulation. We found that ZEB1 is a critical EMT factor for mutant β-catenin-mediated loss of E-cadherin and Claudin-7 in HCT116-P and HCT116-MT cells. We also demonstrated that E-cadherin binds to both WT and mutant β-catenin, and loss of E-cadherin releases β-catenin from the cell membrane and leads to its degradation. Alteration of Claudin-7, as well as both Claudin-7 and E-cadherin respectively caused tight junction (TJ) impairment in HCT116-P, and dual loss of TJs and adherens junctions (AJs) in HCT116-MT. TJ loss increased cell motility, and subsequent AJ loss further up-regulated that. Immunohistochemistry analysis of 101 CRCs revealed high (14.9%), low (52.5%), and undetectable (32.6%) β-catenin nuclear expression, and high β-catenin nuclear expression was significantly correlated with overall survival of CRC patients (P = 0.009). Our findings suggest that β-catenin activation induces EMT progression by modifying cell-cell junctions, and thereby contributes to CRC aggressiveness.ope

Microsatellite instability test using peptide nucleic acid probe-mediated melting point analysis: a comparison study

BACKGROUND: Analysis of high microsatellite instability (MSI-H) phenotype in colorectal carcinoma (CRC) is important for evaluating prognosis and choosing a proper adjuvant therapy. Although the conventional MSI analysis methods such as polymerase chain reaction (PCR) fragment analysis and immunohistochemistry (IHC) show high specificity and sensitivity, there are substantial barriers to their use. METHODS: In this study, we analyzed the MSI detection performance of three molecular tests and IHC. For the molecular tests, we included a recently developed peptide nucleic acid probe (PNA)-mediated real-time PCR-based method using five quasi-monomorphic mononucleotide repeat markers (PNA method) and two conventional PCR fragment analysis methods using NCI markers (NCI method) or five quasi-monomorphic mononucleotide repeat markers (MNR method). IHC analysis was performed with four mismatch repair proteins. The performance of each method was validated in 166 CRC patient samples, which consisted of 76 MSI-H and 90 microsatellite stable (MSS) CRCs previously diagnosed by NCI method. RESULTS: Of the 166 CRCs, 76 MSI-H and 90 MSS CRCs were determined by PNA method. On the other hand, 75 MSI-H and 91 MSS CRCs were commonly determined by IHC and MNR methods. Based on the originally diagnosed MSI status, PNA showed 100% sensitivity and 100% specificity while IHC and MNR showed 98.68% sensitivity and 100% specificity. When we analyzed the maximum sensitivity of MNR and PNA method, which used the same five markers, PNA method could detect alterations in all five mononucleotide repeat markers in samples containing down to 5% MSI-H DNAs, whereas MNR required at least 20% MSI-H DNAs to achieve the same performance. CONCLUSIONS: Based on these findings, we suggest that PNA method can be used as a practical laboratory test for the diagnosis of MSI.ope