C.R. Boland

Metastasis-associated long non-coding RNA drives gastric cancer development and promotes peritoneal metastasis

The prognosis of gastric cancer (GC) patients with peritoneal dissemination remains poor, and a better understanding of the underlying mechanisms is critical for the development of new treatments that will improve survival in these patients. This study aimed to clarify the clinical and biological role of two key metastasis-associated long non-coding RNAs (lncRNAs) in GC. We analyzed the expression levels of two lncRNAs-Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and HOX-Antisense Intergenic RNA (HOTAIR)-by real-time reverse transcription PCR in 300 gastric tissues (150 GC and 150 adjacent normal mucosa), and in seven GC cell lines. Functional characterization for the role of HOTAIR in GC was performed by small interfering RNA (siRNA) knockdown, followed by series of in-vitro and in-vivo experiments. Expression of both lncRNAs was significantly higher in cancerous tissues than in corresponding normal mucosa, and higher expression of these lncRNAs significantly correlated with peritoneal metastasis in GC patients. In addition, elevated HOTAIR expression emerged both as an independent prognostic and risk factor for peritoneal dissemination. SiRNA knockdown of HOTAIR in GC cells significantly inhibited cell proliferation, migration and invasion, but concurrently enhanced the anoikis rate in transfected cells. In an in vivo assay, HOTAIR siRNA-transfected MKN45 cells injected into nude mice inhibited the growth of xenograft tumors and peritoneal metastasis compared with controls. Our data provide novel evidence for the biological and clinical significance of HOTAIR expression as a potential biomarker for identifying patients with peritoneal metastasis, and as a novel therapeutic target in patients with gastric neoplasia.

INFECTION, INFLAMMATION, AND GASTROINTESTINAL CANCER

The struggle to understand the origins of human cancers has captured the imagination of many investigators. The epidemiology of human cancers and the availability of many laboratory models of cancer could give the casual observer the impression that all cancers are a result of exposures to chemical carcinogens in the environment. This is only part of the story, and in most instances environmental exposures are very different in scale from what is required in laboratory animals to induce tumours. Actually, most laboratory models have been carefully developed to match the carcinogen with the host. For example, several alkylating agents can induce intestinal cancers in rodents.1 However, the distribution of the neoplasms throughout the gut varies from one mouse strain to another, and in some instances the carcinogens induce tumours only outside the gut. Lower doses—perhaps those more relevant for human cancers—may be tolerated, and not induce cancer at all. The mechanisms for tumour development in the human gastrointestinal tract appear to be a much more complicated issue. This review of recent advances in basic science will focus on newly discovered mechanisms involved in the development of colorectal cancer (CRC). This is potentially a very broad topic, and therefore we have selected two novel mechanisms for emphasis. Firstly, a virus carried by most healthy individuals has been implicated as a possible cause for chromosomal instability (CIN), the process that leads to aneuploidy. Chromosomal aberrations and this form of genomic instability play a major role in the development and progression of multistep carcinogenesis, such as occurs in CRC.2 Secondly, it is abundantly clear that inflammation is carcinogenic, and furthermore, endogenous processes that can modify the ability of the host to cope with inflammation appear to modify the risk of cancer to the host. There is growing evidence that this may …

Recent insights into the pathogenesis of colorectal cancer.

Purpose of review Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths in the Western world, but our understanding of this disease is incomplete. The recent advent of new technologies has provided novel insights into the pathogenesis of CRC. Recent findings Genome-wide association studies have recently linked CRC to 10 common genetic variants or single-nucleotide polymorphisms that map to chromosomes 8q23, 8q24, 10p14, 11q23, 14q22, 15q13, 16q22, 18q21, 19q13 and 20p1. However, the causal significance of these variants is not understood, and some are located in poorly characterized genomic regions or gene deserts. Recent studies indicate that the single-nucleotide polymorphism rs6983267, which maps to 8q24, serves as an enhancer of MYC expression by binding T cell factor 4 (TCF4) and influencing Wnt signaling. In addition, several microRNAs interact with genes such as K-RAS, APC, p53, PTEN, TCF4, COX-2, DNMT3a and DNMT3b. Germline hypermethylation of the DNA mismatch repair genes MLH1 and MSH2 may serve as predisposing events in some CRC patients. Summary Recent studies have elucidated novel mechanisms involved in CRC, including the involvement of single-nucleotide polymorphisms not located within traditional genes, the role of microRNAs and epimutations in DNA mismatch repair genes. Interestingly, most of this progress has been made by understanding DNA that does not encode genes.

Methylation pattern of the O6‐methylguanine‐DNA methyltransferase gene in colon during progressive colorectal tumorigenesis

O6‐methylguanine‐DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpG sequences within MGMT promoter leads to loss of its protein expression, and if MGMT methylation correlates with G to A transition mutations in KRAS. Two methylation sensitive regions (Mp and Eh region) of MGMT promoter were investigated in 593 specimens of colorectal tissue: 233 CRCs, 104 adenomatous polyps (AP), 220 normal colonic mucosa from CRC patients (N‐C) and 36 normal colonic mucosa specimens obtained from subjects without colorectal neoplasia (N‐N) by combined bisulfite restriction analysis (COBRA). The region‐specific methylation data were compared to the MGMT protein expression, spectrum of KRAS mutations and other clinical features. Extensive (including both Mp and Eh) and partial (either Mp or Eh) MGMT methylation were found in 24.5% and 11.6% of CRCs, 3.8% and 27.9% of APs, 0.5% and 7.7% of C‐Ns and 2.8% and 2.8% of N‐Ns, respectively. Extensive methylation of MGMT promoter was primarily present in CRCs while partial methylation was common in APs. Extensive methylation of MGMT promoter was associated with loss/reduced protein expression (p < 0.0001), as well as with G to A mutations in KRAS (p = 0.0017). We herein provide first evidence that extensive methylation of MGMT promoter region is essential for methylation‐induced silencing of this gene. Our data suggest that MGMT methylation may evolve and spread throughout the promoter in a stepwise manner as the colonic epithelial cells progress through the classical‐adenoma‐cancer multistep cascade.

JC virus infects the enteric glia of patients with chronic idiopathic intestinal pseudo-obstruction

Background and aim: Chronic idiopathic intestinal pseudo-obstruction (CIIP) is characterised by severe impairment of intestinal propulsive motility that mimics bowel obstruction. JC virus (JCV) is a polyomavirus that can infect brain glial cells causing a fatal disease, but may also be found throughout the normal gastrointestinal tract. The hypothesis that JCV infects the myenteric plexuses of patients with CIIP was tested. Methods: 10 patients with CIIP and 61 normal specimens (30 ascending colon and 31 ileum) from patients with uncomplicated colon cancer were studied. DNA was extracted from the myenteric plexuses, and JCV T antigen (TAg) DNA and the viral regulatory region were detected by PCR and sequencing. Immunohistochemistry was performed to detect JCV viral protein expression, neuronal and glial markers. Fluorescence in situ hybridisation was performed for cellular localisation of the JCV infection. Results: Clinical studies demonstrated neurogenic impairment, and pathological analyses showed neuropathy in each patient with CIIP. JCV TAg DNA was found in the myenteric plexuses of 8/10 (80%) of the patients with CIIP and 3/31 (9.7%) of the control patients (p<0.001). All samples were JCV Mad-1 strains. Seven of the 10 CIIP specimens expressed both JCV TAg and the JCV viral protein VP1, while none of the controls expressed either. JCV infection co-localised with glial fibrillary acidic protein expression, a marker of enteric glial cells. Conclusion: JCV infection occurs in the myenteric plexuses of patients with CIIP. The JCV localisation in enteroglial cells suggests a possible pathological role for this virus in enteric neuropathy.

Circulating microRNA-203 predicts prognosis and metastasis in human colorectal cancer.

Background and aims Distant metastasis is a major cause of deaths in patients with colorectal cancer (CRC), which is partly due to lack of robust metastasis-predictive biomarkers. In spite of the important function of microRNA (miR)-203 in cancer metastasis, its clinical significance in CRC metastasis remains unknown. Here, we evaluated the potential role of serum miR-203 as a non-invasive biomarker for CRC metastasis. Methods MiR-203 expression was quantified by quantitative reverse-transcription PCR in 58 pairs of primary CRC (pCRC) and corresponding matched liver metastasis (LM), as well as 186 serum and 154 matched tissue specimens from patients with CRC in cohort 1. Next, we performed validation of miR-203 levels in serum from 144 patients with CRC in an independent cohort (cohort 2). Mouse models of CRC-associated metastases were established to identify the source of circulating miR-203. Expression patterns of miR-203 in tissues were determined by in situ hybridisation. Results MiR-203 expression was significantly upregulated in LM compared with matched pCRC tissues. Serum miR-203 levels were significantly upregulated in a stage-dependent manner, and high miR-203 expression was associated with poor survival in patients with CRC in both patient cohorts. Increased miR-203 levels in serum indicated high risk for poor prognosis (HR=2.1), as well as metastasis to lymph nodes (OR=2.5), liver (OR=6.2), peritoneum (OR=7.2) and distant organs (OR=4.4). Serum miR-203 levels were significantly higher in animals with liver or systemic metastasis compared with controls. Conclusions High levels of serum miR-203 associate with poor survival and metastasis, suggesting it to be a promising non-invasive prognostic and metastasis-predictive biomarker in patients with CRC.

Prevalence of somatic mutl homolog 1 promoter hypermethylation in Lynch syndrome colorectal cancer.

Colorectal cancers (CRCs) that have microsatellite instability (MSI) and mutL homolog 1 (MLH1) immunoloss are observed in 3 clinical scenarios: Lynch syndrome (LS), sporadic MSI CRC, and Lynch‐like syndrome (LLS). v‐Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational analysis is used to differentiate LS from sporadic MSI CRC. The role of MLH1 promoter methylation status for the differential diagnosis of these clinical forms is not well established. The objectives of this study were: 1) to analyze MLH1 promoter methylation in MLH1‐deficient CRCs by pyrosequencing, and 2) to assess its role in the differential diagnosis of MLH1‐deficient CRCs.

Promoter Methylation in the Genesis of Gastrointestinal Cancer

Colorectal cancers (CRC)-and probably all cancers-are caused by alterations in genes. This includes activation of oncogenes and inactivation of tumor suppressor genes (TSGs). There are many ways to achieve these alterations. Oncogenes are frequently activated by point mutation, gene amplification, or changes in the promoter (typically caused by chromosomal rearrangements). TSGs are typically inactivated by mutation, deletion, or promoter methylation, which silences gene expression. About 15% of CRC is associated with loss of the DNA mismatch repair system, and the resulting CRCs have a unique phenotype that is called microsatellite instability, or MSI. This paper reviews the types of genetic alterations that can be found in CRCs and hepatocellular carcinoma (HCC), and focuses upon the epigenetic alterations that result in promoter methylation and the CpG island methylator phenotype (CIMP). The challenge facing CRC research and clinical care at this time is to deal with the heterogeneity and complexity of these genetic and epigenetic alterations, and to use this information to direct rational prevention and treatment strategies.

MiR-320e is a novel prognostic biomarker in colorectal cancer.

Background:Advances in early detection and treatment have improved outcomes in patients with colorectal cancer (CRC). However, there remains a need for robust prognostic and predictive biomarkers. We conducted a systematic discovery and validation of microRNA (miRNA) biomarkers in two clinical trial cohorts of CRC patients.Methods:We performed an initial ‘discovery’ phase using Affymetrix miRNA expression arrays to profile stage III CRC patients with and without tumour recurrence (n=50 per group) at 3-years of follow-up. All patients received adjuvant 5-fluorouracil (5-FU) plus oxaliplatin, that is, FOLFOX, treatment. During ‘validation’, we analysed miRNAs using qRT–PCR in an independent cohort of 237 stage II–IV CRC patients treated with 5-FU-based chemotherapy, as well as in normal colonic mucosa from 20 healthy subjects. Association with disease recurrence, disease-free survival (DFS) and overall survival (OS) was examined using Cox proportional hazard models.Results:In the discovery cohort, miR-320e expression was significantly elevated in stage III colon cancers from patients with vs without recurrence (95% confidence interval (CI)=1.14–1.42; P<0.0001). These results were then independently validated in stage II and III tumours. Specifically, increased miR-320e expression was associated with poorer DFS (hazard ratio (HR)=1.65; 95% CI=1.27–2.13; P=0.0001) and OS (HR=1.78; 95% CI=1.31–2.41; P=0.0003) in stage III CRC patients.Conclusions:In two clinical trial cohorts, a systematic biomarker discovery and validation approach identified miR-320e to be a novel prognostic biomarker that is associated with adverse clinical outcome in stage III CRC patients treated with 5-FU-based adjuvant chemotherapy. These findings have important implications for the personalised management of CRC patients.

Chromosomal instability and cancer: not just one CINgle mechanism.

One of the most impressive undertakings in the life of a somatic cell occurs when it divides into two daughter cells. Nearly 3 billion base pairs of nucleotides packed into 23 pairs of chromosomes are duplicated, line up on a mitotic plate, and are pulled away from their identical sisters, tethered to mitotic spindles. This almost always results in a perfectly symmetrical division of the duplicated genome. Anyone who has dealt with fishing line along the shore of a stream knows how daunting it can be to keep a few metres of monofilament from becoming lethally tangled. Yet, the cell does this nearly every time with ineffable ease and, moreover, detects when the process is misbehaving, and halts progress. When a cell undergoes an unbalanced mitosis, this leads to the creation of aneuploid cells. In spite of the number of mitoses that occur each day, one is hard-pressed to find aneuploid cells in normal tissues. Cancer cells have survival advantages over their normal progenitors, but they tolerate some degree of malfunction in mitotic homeostasis, and are frequently aneuploid. Perhaps even more surprising, the more aneuploidy one finds in a colorectal cancer (CRC) the more deadly the tumour.1 One might anticipate from first principles that a derangement in orderly cell division would be difficult to tolerate, would confer a deficit in fitness, and that mitotic instability would lead to its own demise. However, this is not the case, and understanding this paradox might provide some insight into how cancers develop, and how we might find its Achilles heel. The conceptual challenge for understanding aneuploidy …