Quantitative analysis of ERG expression and its splice isoforms in formalin-fixed, paraffin-embedded prostate cancer samples: association with seminal vesicle invasion and biochemical recurrence.

OBJECTIVES: The proto-oncogene ETS-related gene (ERG) is consistently overexpressed in prostate cancer. Alternatively spliced isoforms of ERG have variable biological activities; inclusion of exon 11 (72 base pairs [bp]) is associated with aggressiveness and progression of disease. Exon 10 (81 bp) has also been shown to be alternatively spliced. Within this study, we assess whether ERG protein, messenger RNA (mRNA), and ERG splice isoform mRNA expression is altered as prostate cancer progresses. METHODS: Detection of the TMPRSS2-ERG fusion was done using direct methods (reverse transcription polymerase chain reaction [PCR] and fluorescence in situ hybridization) and indirect methods for ERG mRNA and protein expression using quantitative PCR and immunohistochemistry, respectively. A linear equation method was used to quantitatively determine relative proportions of ERG variants (ERG72/Δ72, ERG81/Δ81) for each sample. RESULTS: ERG mRNA and protein expression is increased in patients with advanced prostate cancer, with higher levels of ERG expression significantly associated with seminal vesicle invasion (stage pT3b) and biochemical recurrence. Genes involved in cell migration and invasiveness (matrix metalloproteinase 7, osteopontin, and septin 9) are increased in prostate cancers that overexpress ERG. In addition, there is a clear indication of increased retention of exons 10 and 11 in prostate cancer. CONCLUSIONS: Analysis of ERG and its variants may be valuable in determining prognosis and development of prostate cancer.

Conjugated linoleate reduces prostate cancer viability whereas the effects of oleate and stearate are cell line-dependent.

BACKGROUND: In this study, responses to fatty acid treatments in commonly used prostate cancer cell culture models and variability of gene expression between them were determined. MATERIALS AND METHODS: PC3, DU145, LNCaP, VCaP and PNT2 cells were treated with 100 µM of either oleate, stearate or conjugated linoleate. Cell proliferation and viability were assessed using trypan blue and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay respectively. Gene expression was measured using real-time polymerase chain reaction (PCR). RESULTS: Conjugated linoleic acid reduced cell proliferation and viability in all prostate cancer cell lines, whilst the effects of oleic and stearic acid on proliferation were found to be cell line-dependent. A reduction in gene expression of fatty acid desaturases was observed in prostate cancer cell lines compared to normal prostate cells. CONCLUSION: Differential responses of the cell lines investigated here to fatty acid treatment suggest that multiple prostate cancer cell line models should be used when designing experiments aimed at examining lipid metabolism in prostate cancer.

Adaptive changes of the Insig1/SREBP1/SCD1 set point help adipose tissue to cope with increased storage demands of obesity.

The epidemic of obesity imposes unprecedented challenges on human adipose tissue (WAT) storage capacity that may benefit from adaptive mechanisms to maintain adipocyte functionality. Here, we demonstrate that changes in the regulatory feedback set point control of Insig1/SREBP1 represent an adaptive response that preserves WAT lipid homeostasis in obese and insulin-resistant states. In our experiments, we show that Insig1 mRNA expression decreases in WAT from mice with obesity-associated insulin resistance and from morbidly obese humans and in in vitro models of adipocyte insulin resistance. Insig1 downregulation is part of an adaptive response that promotes the maintenance of SREBP1 maturation and facilitates lipogenesis and availability of appropriate levels of fatty acid unsaturation, partially compensating the antilipogenic effect associated with insulin resistance. We describe for the first time the existence of this adaptive mechanism in WAT, which involves Insig1/SREBP1 and preserves the degree of lipid unsaturation under conditions of obesity-induced insulin resistance. These adaptive mechanisms contribute to maintain lipid desaturation through preferential SCD1 regulation and facilitate fat storage in WAT, despite on-going metabolic stress.

A M-MLV reverse transcriptase with reduced RNaseH activity allows greater sensitivity of gene expression detection in formalin fixed and paraffin embedded prostate cancer samples.

Formalin fixed and paraffin embedded (FFPE) human tissue collections are an invaluable resource for retrospective gene expression studies. However formalin fixation results in chemical modification of RNA and increased RNA degradation. This can affect RNA yield and quality. A critical step when analysing gene expression is the conversion of RNA to complementary DNA (cDNA) using a reverse transcriptase (RT) enzyme. FFPE derived RNA may affect the performance and efficiency of the RT enzyme and cDNA synthesis. We directly compared three commonly used FFPE RNA isolation methods and measured RNA yield, purity and integrity. We also assessed the effectiveness of three commercially available Moloney Murine Leukemia Virus (M-MLV) RTs on cDNA synthesis and gene expression sensitivity when using FFPE RNA as a template. Our results show that gene detection sensitivity is dependent on the isolation method, RT and length of the PCR amplicon (<200bp) when using FFPE RNA. The use of an M-MLV RT enzyme with reduced RNaseH activity gave significantly increased qRT-PCR sensitivity when using FFPE RNA derived from prostate tissue. The choice of RT can also affect perceived changes in target gene expression and thus the same RT should be used when attempting to reproduce results from different studies. This study highlights the need to optimise and evaluate RNA isolation methods and RTs when using FFPE RNA as a template in order to maximise a successful outcome in PCR applications.

Epigallocatechin-3-gallate promotes apoptosis and expression of the caspase 9a splice variant in PC3 prostate cancer cells.

Growing evidence suggests that the flavonoid epigallocatechin-3-gallate (EGCG), notably abundant in green tea, has health-promoting properties. We examined the effect of EGCG on cell survival and apoptosis in the prostate cancer cell line PC3. Cell survival was reduced and apoptosis increased significantly with a low dose of 1 µM EGCG. The ability of the anticancer drug cisplatin to promote apoptosis was enhanced by EGCG. Furthermore, EGCG, both alone and in combination with cisplatin, promoted the expression of the pro-apoptotic splice isoform of caspase 9.

Role of splice variants in the metastatic progression of prostate cancer.

AS (alternative splicing) and its role in disease, especially cancer, has come to forefront in research over the last few years. Alterations in the ratio of splice variants have been widely observed in cancer. Splice variants of cancer-associated genes have functions that can alter cellular phenotype, ultimately altering metastatic potential. As metastases are the cause of approximately 90% of all human cancer deaths, it is crucial to understand how AS is dysregulated in metastatic disease. We highlight some recent studies into the relationship between altered AS of key genes and the initiation of prostate cancer metastasis.

An allostatic control of membrane lipid composition by SREBP1.

The maintenance of membrane lipid composition within strict limits is critical to maintain optimum cellular function. The biophysical properties of the membrane can be influenced among other factors by the saturation/unsaturation of the phospholipid fatty acyl chain. The rate-limiting enzyme in unsaturated fatty acid biosynthesis is the desaturase enzyme which in turn is regulated by the lipid transcription factor sterol regulatory element binding protein (SREBP1). In this review, we collect some evidence suggesting SREBP1 network as an important allostatic regulator necessary to maintain the pool of unsaturated fatty acid lipid species that can be incorporated into biological membranes.