MicroRNA-31 modulates tumour sensitivity to radiation in oesophageal adenocarcinoma.

Chemoradiation therapy (CRT) prior to surgery is increasingly the standard of care for locally advanced oesophageal cancer. Radiation therapy is important for local tumour control; however, tumour resistance to radiation is a substantial clinical problem. The mechanism(s) of radioresistance are still poorly understood, however, mounting evidence supports a role for microRNA (miRNA) in modulating key cellular pathways mediating response to radiation. Global miRNA profiling of an established isogenic model of radioresistance in oesophageal adenocarcinoma demonstrated a significant downregulation of miR-31 in radioresistant cells, both basally and in response to radiation. Ectopic re-expression of miR-31 significantly re-sensitised radioresistant cells to radiation. miR-31 was demonstrated to alter the expression of 13 genes involved in DNA repair, which is a critical cellular defence against radiation-induced DNA damage. In oesophageal tumours, miR-31 expression was significantly reduced in patients demonstrating poor histomorphologic response to neoadjuvant CRT, whilst expression of the miR-31-regulated DNA repair genes was significantly increased. Our data suggest a possible mechanism for resistance to CRT, potentially via enhanced DNA repair. This study demonstrates, for the first time, a role for miR-31 in modulating radioresistance and highlights the need for further study investigating the potential role of miR-31 as both a predictive marker of response and a novel therapeutic agent with which to enhance the efficacy of radiation therapy.

Suppression of cancer stemness p21-regulating mRNA and microRNA signatures in recurrent ovarian cancer patient samples.

BACKGROUND: Malignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease. METHODS: Microarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples. RESULTS: Microarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component. CONCLUSION: We present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21 mechanism in ovarian disease. Targeting CSCs within ovarian cancer represents a potential therapeutic avenue.

Inkjet-like printing of single-cells.

Cell sorting and separation techniques are essential tools for cell biology research and for many diagnostic and therapeutic applications. For many of these applications, it is imperative that heterogeneous populations of cells are segregated according to their cell type and that individual cells can be isolated and analysed. We present a novel technique to isolate single cells encapsulated in a picolitre sized droplet that are then deposited by inkjet-like printing at defined locations for downstream genomic analysis. The single-cell-manipulator (SCM) developed for this purpose consists of a dispenser chip to print cells contained in a free flying droplet, a computer vision system to detect single-cells inside the dispenser chip prior to printing, and appropriate automation equipment to print single-cells onto defined locations on a substrate. This technique is spatially dynamic, enabling cell printing on a wide range of commonly used substrates such as microscope slides, membranes and microtiter plates. Demonstration experiments performed using the SCM resulted in a printing efficiency of 87% for polystyrene microbeads of 10 µm size. When the SCM was applied to a cervical cancer cell line (HeLa), a printing efficiency of 87% was observed and a post-SCM cell viability rate of 75% was achieved.

BRAFV600E: implications for carcinogenesis and molecular therapy.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway is frequently mutated in human cancer. This pathway consists of a small GTP protein of the RAS family that is activated in response to extracellular signaling to recruit a member of the RAF kinase family to the cell membrane. Active RAF signals through MAP/ERK kinase to activate ERK and its downstream effectors to regulate a wide range of biological activities including cell differentiation, proliferation, senescence, and survival. Mutations in the v-raf murine sarcoma viral oncogenes homolog B1 (BRAF) isoform of the RAF kinase or KRAS isoform of the RAS protein are found as activating mutations in approximately 30% of all human cancers. The BRAF pathway has become a target of interest for molecular therapy, with promising results emerging from clinical trials. Here, the role of the most common BRAF mutation BRAF(V600E) in human carcinogenesis is investigated through a review of the literature, with specific focus on its role in melanoma, colorectal, and thyroid cancers and its potential as a therapeutic target.

Regulation of microRNA biosynthesis and expression in 2102Ep embryonal carcinoma stem cells is mirrored in ovarian serous adenocarcinoma patients.

BACKGROUND: Tumours with high proportions of differentiated cells are considered to be of a lower grade to those containing high proportions of undifferentiated cells. This property may be linked to the differentiation properties of stem cell-like populations within malignancies. We aim to identify molecular mechanism associated with the generation of tumours with differing grades from malignant stem cell populations with different differentiation potentials. In this study we assessed microRNA (miRNA) regulation in two populations of malignant Embryonal Carcinoma (EC) stem cell, which differentiate (NTera2) or remain undifferentiated (2102Ep) during tumourigenesis, and compared this to miRNA regulation in ovarian serous carcinoma (OSC) patient samples. METHODS: miRNA expression was assessed in NTera2 and 2102Ep cells in the undifferentiated and differentiated states and compared to that of OSC samples using miRNA qPCR. RESULTS: Our analysis reveals a substantial overlap between miRNA regulation in 2102Ep cells and OSC samples in terms of miRNA biosynthesis and expression of mature miRNAs, particularly those of the miR-17/92 family and clustering to chromosomes 14 and 19. In the undifferentiated state 2102Ep cells expressed mature miRNAs at up to 15,000 fold increased levels despite decreased expression of miRNA biosynthesis genes Drosha and Dicer. 2102Ep cells avoid differentiation, which we show is associated with consistent levels of expression of miRNA biosynthesis genes and mature miRNAs while expression of miRNAs clustering to chromosomes 14 and 19 is deemphasised. OSC patient samples displayed decreased expression of miRNA biosynthesis genes, decreased expression of mature miRNAs and prominent clustering to chromosome 14 but not 19. This indicates that miRNA biosynthesis and levels of miRNA expression, particularly from chromosome 14, are tightly regulated both in progenitor cells and in tumour samples. CONCLUSION: miRNA biosynthesis and expression of mature miRNAs, particularly the miR-17/92 family and those clustering to chromosomes 14 and 19, are highly regulated in both progenitor cells and tumour samples. Strikingly, 2102Ep cells are not simply malfunctioning but respond to differentiation specifically, a mechanism that is highly relevant to OSC samples. Our identification and future manipulation of these miRNAs may facilitate generation of lower grade malignancies from these high-grade cells.

Gene expression analysis of diagnostic biopsies predicts pathological response to neoadjuvant chemoradiotherapy of esophageal cancer.

OBJECTIVE: This study explored gene expression differences in predicting response to chemoradiotherapy in esophageal cancer. PURPOSE: A major pathological response to neoadjuvant chemoradiation is observed in about 40% of esophageal cancer patients and is associated with favorable outcomes. However, patients with tumors of similar histology, differentiation, and stage can have vastly different responses to the same neoadjuvant therapy. This dichotomy may be due to differences in the molecular genetic environment of the tumor cells. BACKGROUND DATA: Diagnostic biopsies were obtained from a training cohort of esophageal cancer patients (13), and extracted RNA was hybridized to genome expression microarrays. The resulting gene expression data was verified by qRT-PCR. In a larger, independent validation cohort (27), we examined differential gene expression by qRT-PCR. The ability of differentially-regulated genes to predict response to therapy was assessed in a multivariate leave-one-out cross-validation model. RESULTS: Although 411 genes were differentially expressed between normal and tumor tissue, only 103 genes were altered between responder and non-responder tumor; and 67 genes differentially expressed >2-fold. These included genes previously reported in esophageal cancer and a number of novel genes. In the validation cohort, 8 of 12 selected genes were significantly different between the response groups. In the predictive model, 5 of 8 genes could predict response to therapy with 95% accuracy in a subset (74%) of patients. CONCLUSIONS: This study has identified a gene microarray pattern and a set of genes associated with response to neoadjuvant chemoradiation in esophageal cancer. The potential of these genes as biomarkers of response to treatment warrants further investigation.

Potentially important microRNA cluster on chromosome 17p13.1 in primary peritoneal carcinoma.

MicroRNAs are a group of small non-coding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature microRNAs in human cancers. We characterized the alteration in expression of a select group of microRNAs in primary peritoneal carcinoma relative to matched cases of ovarian serous carcinoma. MicroRNA expression was analysed using semi-quantitative stem-loop RT-PCR on a set of 34 formalin-fixed paraffin-embedded samples. Protein expression of p53 and bcl-2 was quantified in the corresponding tissue microarray. We provide definitive evidence that there is downregulation of a select group of microRNAs in tumours meeting Gynaecological Oncology Group criteria for primary peritoneal carcinoma relative to ovarian serous carcinoma. Specifically, we show decreased p53 expression and downregulation of miR-195 and miR-497 from the microRNA cluster site at chromosome 17p13.1 in primary peritoneal carcinoma relative to ovarian serous carcinoma. miR-195 and miR-497 may have potential roles as tumour-suppressor genes in primary peritoneal tumourigenesis.

Geographical mapping of a multifocal thyroid tumour using genetic alteration analysis & miRNA profiling.

BACKGROUND: Papillary thyroid carcinoma (PTC) frequently presents as multiple tumour-foci within a single thyroid gland or pluriform, with synchronous tumours comprising different histological variants, raising questions regarding its clonality. Among the genetic aberrations described in PTC, the BRAF V600E mutation and ret/PTC activation occur most commonly. Several studies have investigated the genetic alteration status of multifocal thyroid tumours, with discordant results. To address the question of clonality this study examined disparate geographical and morphological areas from a single PTC (classic PTC, insular and anaplastic foci, and tumour cells adjacent to vascular invasion and lymphocytic infiltrate) for the presence of ret/PTC 1 or BRAF mutations. Moreover, we wanted to investigate the consistency of miRNA signatures within disparate areas of a tumour, and geographical data was further correlated with expression profiles of 330 different miRNAs. Putative miRNA gene targets were predicted for differentially regulated miRNAs and immunohistochemistry was performed on tissue sections in an effort to investigate phenotypic variations in microvascular density (MVD), and cytokeratin and p53 protein expression levels. RESULTS: All of the morphological areas proved negative for ret/PTC 1 rearrangement. Two distinct foci with classic morphology harboured the BRAF mutation. All other regions, including the insular and anaplastic areas were negative for the mutation. MiRNA profiles were found to distinguish tumours containing the BRAF mutation from the other tumour types, and to differentiate between the more aggressive insular & anaplastic tumours, and the classic variant. Our data corroborated miRNAs previously discovered in this carcinoma, and additional miRNAs linked to various processes involved in tumour growth and proliferation. CONCLUSION: The initial genetic alteration analysis indicated that pluriform PTC did not necessarily evolve from classic PTC progenitor foci. Analysis of miRNA profiles however provided an interesting variation on the clonality question. While hierarchical clustering analysis of miRNA expression supported the hypothesis that discrete areas did not evolve from clonal expansion of tumour cells, it did not exclude the possibility of independent mutational events suggesting both phenomena might occur simultaneously within a tumour to enhance cancer progression in geographical micro-environments within a tumour.

Altered eIF6 and Dicer expression is associated with clinicopathological features in ovarian serous carcinoma patients.

MicroRNAs are a group of small non-coding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature microRNAs in human cancers. Production and function of microRNAs require coordinated processing by proteins of the microRNA machinery. Dicer and Drosha (RNase III endonucleases) are essential components of the microRNA machinery. Recently, the ribosome anti-association factor eIF6 has also been found to have a role in microRNA-mediated post-transcriptional silencing. We characterized the alterations in the expression of genes encoding proteins of microRNA machinery in ovarian serous carcinoma. Protein expression of eIF6 and Dicer was quantified in a tissue microarray of 66 ovarian serous carcinomas. Dicer, Drosha and eIF6 mRNA expression was analysed using quantitative reverse transcription-PCR on an independent set of 50 formalin-fixed, paraffin-embedded ovarian serous carcinoma samples. Expression profiles of eIF6 and Dicer were correlated with clinicopathological and patient survival data. We provide definitive evidence that eIF6 and Dicer are both upregulated in a significant proportion of ovarian serous carcinomas and are associated with specific clinicopathological features, most notably low eIF6 expression being associated with reduced disease-free survival. The status of eIF6 and proteins of the microRNA machinery may help predict toxicity and susceptibility to future interfering RNA-based therapy.

p16(INK4A) genetic and epigenetic profiles differ in relation to age and site in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) typically affects male smokers older than 55 years. Recently, an increase in the incidence of HNSCC in young adults has been recognized, many of them nonsmokers and females. Functional inactivation of p16 is known to be a common event in HNSCC, mainly by either deletion or methylation. A previous study by this group has shown that p16 deletions in HNSCC are significantly associated with age. The primary objective of this study was to evaluate additional molecular alterations of p16 in HNSCC, specifically in relation to age, site, and human papillomavirus (HPV) status. Patients ranging in age from 22 to 76 years with HNSCC were prospectively identified (n = 24). Methylation-specific polymerase chain reaction and immunohistochemistry were used to evaluate p16 gene inactivation and p16 protein expression, respectively. HPV 16 status was determined for each case. Overall, p16 inactivation was a frequent event detected in 46% of cases. Methylation of p16 was more often detected in females than males (P = .05). All cases showing p16 methylation were from the anterior tongue, and 75% of them were young patients. The results indicate that p16 methylation is a more common event in those younger than 40 years in contrast to p16 deletions, which are more common in those older than 40 years. Consequently, it appears that specific modes of inactivation of p16 in HNSCC are related to specific patient risk profiles. Interestingly, HPV 16 messenger RNA was detected exclusively in HNSCC from the base of tongue lesions and was only found in males. This differs from the patient profile of HNSCC in the young, which affects the anterior tongue and commonly females, thus, making it highly unlikely that this virus is a primary causative agent of HNSCC in these young adults.

A molecular expression signature distinguishing follicular lesions in thyroid carcinoma using preamplification RT-PCR in archival samples.

Follicular variant of papillary thyroid carcinoma is a lesion that frequently causes difficulties from a diagnostic perspective in the laboratory. The purpose of this study was to interrogate a cohort of archival thyroid lesions using gene expression analysis of a panel of markers proposed to have utility as adjunctive markers in the diagnosis of thyroid neoplasia and follicular variant of papillary thyroid carcinoma in particular. Laser Capture Microdissection was used to procure pure cell populations for extraction. In addition a novel, multiplex preamplification technique was used to facilitate analysis of multiple targets. The panel comprised: HLA-DMA, HLA-DBQ1, CD74, CSNK1G2, IRF3, KRAS2, LYN, MT1K, MT1X, RAB23, TGFB1 and TOP2A, with CDKN1B as an endogenous control. Expression profiles for each target were generated using TaqMan Real-Time PCR. HLA-DMA, HLA-DQB1, MT1X, CSNK1G2 and RAB23 were found to be differentially expressed (P<0.05) when comparing follicular adenoma and follicular variant of papillary thyroid carcinoma. Comparison of follicular adenoma and follicular thyroid carcinoma groups showed significant differential expression for MT1K, MT1X and RAB23 (P<0.05). Comparison of the papillary thyroid carcinoma group (classic and follicular variants) and the follicular adenoma group showed differential expression for CSNK1G2, HLA-DQB1, MT1X and RAB23 (P<0.05). Finally, KRAS2 was found to be differentially expressed (P<0.05) when comparing the papillary thyroid carcinoma and follicular thyroid carcinoma groups. This panel of molecular targets discriminates between follicular adenoma, papillary thyroid carcinoma, follicular variant of papillary thyroid carcinoma and follicular thyroid carcinoma by their expression repertoires. It may have utility for broader use in the setting of fine-needle aspiration cytology and could improve the definitive diagnosis of certain categories of thyroid malignancy.

Detection of a tyrosine phosphatase LAR on intestinal epithelial cells and intraepithelial lymphocytes in the human duodenum.

Studies of tyrosine phosphorylation in the human duodenum have indicated that proliferating cells in the middle portion of the duodenal crypt were devoid of this feature, suggesting that tyrosine kinase activation is not a dominant factor in crypt cell proliferation, and that consequently tyrosine phosphatase activity may be a more critical factor in crypt cell development. We investigated the expression of the leukocyte common antigen-related receptor (LAR) family of tyrosine phosphatases. A flow cytometry system was used to examine cells from the surface, mid-portion, and lower part of the crypt. Individual cell populations were immunostained with anti-LAR antibodies using phycoerythrin-conjugated anti-CD3 to discriminate between epithelial cells (CD3-) and intraepithelial lymphocytes (CD3+). Epithelial cells expressed LAR throughout the crypt. Expression of LAR was maximal in the mid-portion of the crypt with lower expression at the top of the villi. Intraepithelial lymphocytes expressed low levels of LAR at the tips of the villi with stronger expression extending towards the base of the crypt. These findings were confirmed by immunohistochemistry on paraffin-fixed sections. Of note, peripheral blood lymphocytes expressed less LAR than IEL. These observations suggest the possibility that tyrosine phosphatase LAR may be of importance in the regulation of crypt cell proliferation. Moreover, as the extracellular domain of LAR has homology with adhesion molecules, the finding of this molecule on IEL could suggest a possible functional role in homing of this unique lymphocyte.