Comparative Analysis of miRNA and EMT Markers in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most commonly diagnosed malignant condition in the world. Micro RNAs (miRNAs) as well as epithelial to mesenchymal transition (EMT) play an important role in the pathogenesis of CRC. We performed a comparative analysis of the expression of selected miRNA genes and EMT markers in bioptic samples from patients (n = 45) with primary CRC or metastatic (m)CRC to the regional lymph node using reverse transcription-quantitative PCR and IHC staining. Results: Out of all miRNA analyzed, the miR-17 expression was most significantly different and associated with lower risk of CRC spread to the lymph node. In addition, significant relationships were found between the tumor side localization and several miRNAs expressions (miR-9, miR-29b, miR-19a, miR-19b, miR-21, miR-106a, miR-20a and miR-17). In addition, of the examined EMT markers, only VEGFA expression correlated with tumor progression (tumor grade G2). In the examined set of patient samples and their matched healthy tissue, several specific molecular markers (miRNAs associated with EMT and tumor progression) were identified with a promising prognostic potential. Their further examination in larger patient cohorts is planned to validate the present data.

Distant Metastasis in Colorectal Cancer Patients-Do We Have New Predicting Clinicopathological and Molecular Biomarkers? A Comprehensive Review.

Colorectal cancer (CRC) remains a serious health problem worldwide. Approximately half of patients will develop distant metastasis after CRC resection, usually with very poor prognosis afterwards. Because patient performance after distant metastasis surgery remains very heterogeneous, ranging from death within 2 years to a long-term cure, there is a clinical need for a precise risk stratification of patients to aid pre- and post-operative decisions. Furthermore, around 20% of identified CRC cases are at IV stage disease, known as a metastatic CRC (mCRC). In this review, we overview possible molecular and clinicopathological biomarkers that may provide prognostic and predictive information for patients with distant metastasis. These may comprise sidedness of the tumor, molecular profile and epigenetic characteristics of the primary tumor and arising metastatic CRC, and early markers reflecting cancer cell resistance in mCRC and biomarkers identified from transcriptome. This review discusses current stage in employment of these biomarkers in clinical practice as well as summarizes current experience in identifying predictive biomarkers in mCRC treatment.

Contribution of in vitro comparison of colorectal carcinoma cells from primary and metastatic lesions to elucidation of mechanisms of tumor progression and response to anticancer therapy.

Colorectal cancer has been a leading cause of cancer-related morbidity and mortality. For the research and individualization of therapy, primary cell lines of the colorectal cancer appear to be still an invaluable tool. We evaluated the differences in metastatic potential between four isolated primary colon cancer cells and cells derived from their lymph node metastasis. These results were compared with correspond immortalized cells-SW480 and SW620, respectively. The ability to migrate was tested using real-time measurement in xCELLigence system. Expressions of molecules involved in adhesion and invasion processes were examined using RT-PCR and western blot analysis. Furthermore, impact of cytotoxic effect of selected chemotherapeutics (irinotecan, oxaliplatin) and biological therapy (bevacizumab, cetuximab, panitumumab) was assessed by the WST assay. As expected, cell lines derived from lymph node migrated more aggressively and higher expression of adhesion molecules ICAM-1, EpCAM, and N-cadherin was detected. The expression of MMP-2 and -9 was elevated, on the other hand, in cell lines derived from primary tumor cancer cells as well as the expression of miR-21, miR-29a, and miR-200a. The most pronounced cytotoxic effect has been recorded with oxaliplatin and irinotecan (IC50 = 48.23 resp. 0.11 µg/ml), especially in cells originating from lymph node metastases. In total, comparison of isolated cell lines and immortalized cell lines has shown many similarities, as well as several differences. Adhesion/invasion molecules and several miRNAs, which play an important role in tumor development and the invasive and migratory behavior, could be a useful therapeutic target in malignant colorectal cancer.

Aberrant methylation of tumour suppressor genes WT1, GATA5 and PAX5 in hepatocellular carcinoma.

BACKGROUND: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. METHODS: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. RESULTS: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient's age at the time of diagnosis. CONCLUSIONS: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

Hypersensitivity to chemoradiation in FANCA carrier with cervical carcinoma-A case report and review of the literature.

OBJECTIVE: Compared to Fanconi anemia (FA) patients with homozygous defective two-alleles inheritance, there is a scarce or no evidence on one defective allele FANCA carriers, with respect to their cancer incidence, clinical and in vitro radiosensitivity and chemosensitivity. On that account, we report a case of a 30-year old FANCA mutation carrier woman with uterine cervix adenocarcinoma who was treated with chemoradiotherapy, in which unexpected acute toxicity and fatal late morbidity occured. METHODS: We also report the results of an in vitro test for radiosensitivity, immunohistochemical examination with FANCA staining and human papillomavirus genotypization, and a review of the literature for FA carrier patients with respect to cancer incidence, clinical and in vitro response to chemo/radiotherapy, options of early heterozygosity detection, and methods of in vitro prediction of hypersensitivity to oncologic treatment. CONCLUSION: Although there are no standard guidelines for management of FA carriers with malignancies and reports about chemo- or radiosensitivity in this population are scarce; patients with FA-A heterozygosity may have a high rate of complications from chemo/radiotherapy. Up to now, an optimum method for the prediction of radiosensitivity and the best parameter has not been found. Clinical radioresponsiveness is unpredictable in FA carriers and there is a pressing need of new rapid and predictive in vitro assays of radiation responses. Until then, the treatment of FA carriers with malignancies should be individualized, with respect to potential hypersensitivity to ionizing radiation or cross-linking agents.

Differences in genome, transcriptome, miRNAome, and methylome in synchronous and metachronous liver metastasis of colorectal cancer.

Despite distant metastases being the critical factor affecting patients' survival, they remain poorly understood. Our study thus aimed to molecularly characterize colorectal cancer liver metastases (CRCLMs) and explore whether molecular profiles differ between Synchronous (SmCRC) and Metachronous (MmCRC) colorectal cancer. This characterization was performed by whole exome sequencing, whole transcriptome, whole methylome, and miRNAome. The most frequent somatic mutations were in APC, SYNE1, TP53, and TTN genes. Among the differently methylated and expressed genes were those involved in cell adhesion, extracellular matrix organization and degradation, neuroactive ligand-receptor interaction. The top up-regulated microRNAs were hsa-miR-135b-3p and -5p, and the hsa-miR-200-family while the hsa-miR-548-family belonged to the top down-regulated. MmCRC patients evinced higher tumor mutational burden, a wider median of duplications and deletions, and a heterogeneous mutational signature than SmCRC. Regarding chronicity, a significant down-regulation of SMOC2 and PPP1R9A genes in SmCRC compared to MmCRC was observed. Two miRNAs were deregulated between SmCRC and MmCRC, hsa-miR-625-3p and has-miR-1269-3p. The combined data identified the IPO5 gene. Regardless of miRNA expression levels, the combined analysis resulted in 107 deregulated genes related to relaxin, estrogen, PI3K-Akt, WNT signaling pathways, and intracellular second messenger signaling. The intersection between our and validation sets confirmed the validity of our results. We have identified genes and pathways that may be considered as actionable targets in CRCLMs. Our data also provide a valuable resource for understanding molecular distinctions between SmCRC and MmCRC. They have the potential to enhance the diagnosis, prognostication, and management of CRCLMs by a molecularly targeted approach.

The dynamics of telomere length in primary and metastatic colorectal cancer lesions.

Telomeric sequences, the structures comprised of hexanucleotide repeats and associated proteins, play a pivotal role in chromosome end protection and preservation of genomic stability. Herein we address telomere length (TL) dynamics in primary colorectal cancer (CRC) tumour tissues and corresponding liver metastases. TL was measured by multiplex monochrome real-time qPCR in paired samples of primary tumours and liver metastases along with non-cancerous reference tissues obtained from 51 patients diagnosed with metastatic CRC. Telomere shortening was observed in the majority of primary tumour tissues compared to non-cancerous mucosa (84.1%, p < 0.0001). Tumours located within the proximal colon had shorter TL than those in the rectum (p < 0.05). TL in liver metastases was not significantly different from that in primary tumours (p = 0.41). TL in metastatic tissue was shorter in the patients diagnosed with metachronous liver metastases than in those diagnosed with synchronous liver metastases (p = 0.03). The metastatic liver lesions size correlated with the TL in metastases (p < 0.05). Following the neoadjuvant treatment, the patients with rectal cancer had shortened telomeres in tumour tissue than prior to the therapy (p = 0.01). Patients with a TL ratio between tumour tissue and the adjacent non-cancerous mucosa of ≥ 0.387 were associated with increased overall survival (p = 0.01). This study provides insights into TL dynamics during progression of the disease. The results show TL differences in metastatic lesions and may help in clinical practice to predict the patient's prognosis.

Combined Therapy of Locally Advanced Oesophageal and Gastro-Oesophageal Junction Adenocarcinomas: State of the Art and Aspects of Predictive Factors.

The following main treatment approaches are currently used in locally advanced adenocarcinomas of the oesophagus and gastrooesophageal junction (GOJ): preoperative chemoradiotherapy and surgery, and perioperative chemotherapy and surgery. While preoperative chemoradiotherapy is used primarily in oesophageal tumours, perioperative chemotherapy is the treatment of choice in Western countries for gastric cancer. The optimal treatment strategy for GOJ adenocarcinoma is still not clear. In comparison to other malignancies, biomarkers are used as predictive factors in distal oesophageal and GOJ adenocarcinomas in a very limited way, and moreover, only in metastatic stages (e.g., HER2 status, or microsatellite instability status). The aim of the article is to provide an overview of current treatment options in locally advanced adenocarcinomas of oesophagus and GOJ based on the latest evidence, including the possible potential of predictive biomarkers in optimizing treatment.

Zinc alters cytoskeletal integrity and migration in colon cancer cells.

Zinc has been shown to have inhibitory effects on proliferation and metabolism of malignant colonocytes. Still, there is no information available concerning putative effects of zinc against motility and migration of colon cancer cells. Using fluorescence microscopy, immunoblotting and microflorimetry we show that treatment with zinc sulfate affected motility, invasiveness, cytoskeletal integrity and expression of selected markers (E-cadherin, catenin, vimentin, tubulin and actin) of invasive SW480 colon tumor cells. These results emphasize the possible multitudinous role of zinc in the process of colon cancer development and hint at the potential of this element in chemoprevention of advanced colorectal carcinoma.

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 (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.

Irinotecan induces senescence and apoptosis in colonic cells in vitro.

Irinotecan (CPT-11) is topoisomerase I inhibitor used in the treatment of disseminated colorectal cancer. In colon cancer cells it induces DNA damage which leads to cytotoxicity with ensuing apoptosis or premature senescence. Despite its clinical use and efficiency in malignant colonocytes, its effects in normal colonic cells are relatively underexplored. In this work we report that CPT-11 induces dose-dependent cytotoxicity which results in apoptosis and premature senescence whose occurrence nevertheless varies in relation to the type of exposed cells. In normal colonic epithelial cells (NCM) the prevailing type of response is apoptosis whereas in normal colonic fibroblasts (NCF) it is premature senescence. Further analyses showed that CPT-11 induced in both types of cells DNA damage and activated stress response pathways including p53 and p16 but with varying activity of stress kinase p38 and selected stress-associated microRNAs. Epithelial cells upregulated the expression of p53, which was subsequently specifically phosphorylated, massively activated p38 and initiated mitochondrial, caspase-dependent apoptosis. These events occurred in the presence of moderately increased expression of miR-34a only. Conversely, in colonic fibroblasts p38 was only moderately activated, p53 as well as p16 expressions were upregulated in the presence of increased expression of miR-34a, miR-128a and miR-449a. Caspase-dependent apoptosis was found only in a minority of treated cells and the premature senescence phenotype was prevailing. Specific inhibition further proved that p53-dependent as well as independent mechanisms might be responsible for these cell type-specific differences.

Diverse sensitivity of cells representing various stages of colon carcinogenesis to increased extracellular zinc: implications for zinc chemoprevention.

The relationship between zinc intake and risk of colon cancer is widely recognized. Despite reported mechanisms of zinc-mediated effects in colonic cells no information is available on whether zinc is capable of inducing cell death of malignant colonocytes. The present study shows that increased external zinc concentrations inhibit cell growth of three different colon cancer cell lines representing different stages of colon cancer: HCT-116, HT-29 and SW620 cells and induce their death. Of the tested cell lines, SW620 cells proved to be the most sensitive to externally added zinc and this sensitivity was at least partly due to increased levels of intracellular free zinc and the inability to overexpress metallothionein. Further studies into the mechanisms of zinc-induced cell injury and cell death revealed oxidative stress as the most important underlying mechanism activating stress kinase-dependent signaling, perturbation of mitochondria and plasma membrane damage. In addition, observed cell death in individual cell populations was cell line-dependent and variable including cells displaying features of apoptosis, necrosis, autophagy and other mixed-types. In conclusion, presented results for the first time show variability of responses to zinc in colon cancer at different stages as modeled in vitro and suggest that zinc-induced cell death despite common underlying mechanism(s) might have a variable nature.