Richard Flavin

Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells

BackgroundArchival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens.ResultsOur results show that miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR. The levels of expression of miRNA detected in total RNA extracted from FFPE were higher than that extracted from snap frozen cells when the quantity of total RNA was identical. This phenomenon is most likely explained by the fact that larger numbers of FFPE cells were required to generate equivalent quantities of total RNA than their snap frozen counterparts.ConclusionWe hypothesise that methylol cross-links between RNA and protein which occur during tissue processing inhibit the yield of total RNA. However, small RNA molecules appear to be less affected by this process and are recovered more easily in the extraction process. In general miRNAs demonstrated reliable expression levels in FFPE compared with snap frozen paired samples, suggesting these molecules might prove to be robust targets amenable to detection in archival material in the molecular pathology setting.

Fatty acid synthase as a potential therapeutic target in cancer

Fatty acid synthase (FASN) is a key enzyme involved in neoplastic lipogenesis. Overexpression of FASN is common in many cancers, and accumulating evidence suggests that it is a metabolic oncogene with an important role in tumor growth and survival, making it an attractive target for cancer therapy. Early small-molecule FASN inhibitors such as cerulenin, C75 and orlistat have been shown to induce apoptosis in several cancer cell lines and to induce tumor growth delay in several cancer xenograft models but their mechanism is still not well understood. These molecules suffer from pharmacological limitations and weight loss as a side effect that prevent their development as systemic drugs. Several potent inhibitors have recently been reported that may help to unravel and exploit the full potential of FASN as a target for cancer therapy in the near future. Furthermore, novel sources of FASN inhibitors, such as green tea and dietary soy, make both dietary manipulation and chemoprevention potential alternative modes of therapy in the future.

Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors

Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial–stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.

MYC regulation of a "poor-prognosis" metastatic cancer cell state

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different “poor-outcome” cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by “poor-prognosis” signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

Metabolic alterations and targeted therapies in prostate cancer

Cancer cells synthesize de novo large amounts of fatty acids and cholesterol, irrespective of the circulating lipid levels and benefit from this increased lipid synthesis in terms of growth advantage, self‐survival and drug resistance. Key lipogenic alterations that commonly occur in prostate cancer include over‐expression of the enzyme fatty acid synthase (FASN) and deregulation of the 5‐AMP‐activated protein kinase (AMPK). FASN is a key metabolic enzyme that catalyses the synthesis of palmitate from the condensation of malonyl‐CoA and acetyl‐CoA de novo and plays a central role in energy homeostasis, by converting excess carbon intake into fatty acids for storage. AMPK functions as a central metabolic switch that governs glucose and lipid metabolism. Recent interest has focused on the potential of targeting metabolic pathways that may be altered during prostate tumorigenesis and progression. Several small molecule inhibitors of FASN have now been described or in development for therapeutic use; in addition, drugs that directly or indirectly induce AMPK activation have potential benefit in prostate cancer prevention and treatment. Copyright

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.

Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials.

BackgroundArchival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp).ResultsResults from the Bioanalyzer and TaqMan® data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70°C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan® PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification.ConclusionModification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan® PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.

p16INK4A 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.

Analysis of the 10q11 Cancer Risk Locus Implicates MSMB and NCOA4 in Human Prostate Tumorigenesis

Genome-wide association studies (GWAS) have established a variant, rs10993994, on chromosome 10q11 as being associated with prostate cancer risk. Since the variant is located outside of a protein-coding region, the target genes driving tumorigenesis are not readily apparent. Two genes nearest to this variant, MSMB and NCOA4, are strong candidates for mediating the effects of rs109939934. In a cohort of 180 individuals, we demonstrate that the rs10993994 risk allele is associated with decreased expression of two MSMB isoforms in histologically normal and malignant prostate tissue. In addition, the risk allele is associated with increased expression of five NCOA4 isoforms in histologically normal prostate tissue only. No consistent association with either gene is observed in breast or colon tissue. In conjunction with these findings, suppression of MSMB expression or NCOA4 overexpression promotes anchorage-independent growth of prostate epithelial cells, but not growth of breast epithelial cells. These data suggest that germline variation at chromosome 10q11 contributes to prostate cancer risk by influencing expression of at least two genes. More broadly, the findings demonstrate that disease risk alleles may influence multiple genes, and associations between genotype and expression may only be observed in the context of specific tissue and disease states.

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.