Characterization of farnesyl diphosphate farnesyl transferase 1 (FDFT1) expression in cancer.

AIM: To help characterize the FDFT1 gene and protein expression in cancer. Cholesterol represents an important structural component of lipid rafts. These specializations can be involved in pathways stimulating cell growth, survival and other processes active in cancer. This cellular compartment can be expanded by acquisition of cholesterol from the circulation or by its synthesis in a metabolic pathway regulated by the FDFT1 enzyme. Given the critical role this might play in carcinogenesis and in the behavior of cancers, we have examined the level of this enzyme in various types of human cancer. Our demonstration of elevated levels of FDFT1 mRNA and protein in some tumors relative to surrounding normal tissue identifies this as a possible biomarker for disease development and progression, and as a potential new target for the treatment of cancer.

Update on the clinical utility of an RNA interference-based treatment: focus on Patisiran.

RNA interference (RNAi) is a naturally existing endogenous mechanism for post-transcriptional gene regulation, nowadays commonly utilized for functional characterization of genes and development of potential treatment strategies for diseases. RNAi-based studies for therapy, after being examined for over a decade, are finally in the pipeline for developing a potential treatment for the mutated transthyretin (TTR) gene, which gives rise to a dysfunctional TTR protein. This dysfunctional protein causes TTR amyloidosis (ATTR), an inherited, progressively incapacitating, and often fatal genetic disorder. TTR is a protein produced in the liver, and functions as a carrier for retinol-binding protein and also thyroxine. This protein facilitates the transport of vitamin A around the human body. A mutation or misprint in the code of this protein results in an abnormal folding of the protein. Therefore, not only does the transportation of the vitamin A become disabled, but also there will be formation of clusters called amyloid deposits, which attack the heart and the nerves causing some patients to be unconditionally bound to bed. ATTR is a hereditary autosomal dominant disease with a 50% chance of inheritance by offspring, even with just one of the parents having a single defective allele of this gene. Alnylam Pharmaceuticals worked on the concept of RNAi therapy for years, which led to the introduction of lipid nanoparticles encircling small interfering RNAs. The drug showed extremely positive results since the first trial, and a great percentage of defective protein reduction. This drug was later named Patisiran.

Target-based drug discovery for [Formula: see text]-globin disorders: drug target prediction using quantitative modeling with hybrid functional Petri nets.

Recent molecular studies provide important clues into treatment of [Formula: see text]-thalassemia, sickle-cell anaemia and other [Formula: see text]-globin disorders revealing that increased production of fetal hemoglobin, that is normally suppressed in adulthood, can ameliorate the severity of these diseases. In this paper, we present a novel approach for drug prediction for [Formula: see text]-globin disorders. Our approach is centered upon quantitative modeling of interactions in human fetal-to-adult hemoglobin switch network using hybrid functional Petri nets. In accordance with the reverse pharmacology approach, we pose a hypothesis regarding modulation of specific protein targets that induce [Formula: see text]-globin and consequently fetal hemoglobin. Comparison of simulation results for the proposed strategy with the ones obtained for already existing drugs shows that our strategy is the optimal as it leads to highest level of [Formula: see text]-globin induction and thereby has potential beneficial therapeutic effects on [Formula: see text]-globin disorders. Simulation results enable verification of model coherence demonstrating that it is consistent with qPCR data available for known strategies and/or drugs.

Validation of signalling pathways: Case study of the p16-mediated pathway.

p16 is recognized as a tumor suppressor gene due to the prevalence of its genetic inactivation in all types of human cancers. Additionally, p16 gene plays a critical role in controlling aging, regulating cellular senescence, detection and maintenance of DNA damage. The molecular mechanism behind these events involves p16-mediated signaling pathway (or p16- Rb pathway), the focus of our study. Understanding functional dependence between dynamic behavior of biological components involved in the p16-mediated pathway and aforesaid molecular-level events might suggest possible implications in the diagnosis, prognosis and treatment of human cancer. In the present work, we employ reverse-engineering approach to construct the most detailed computational model of p16-mediated pathway in higher eukaryotes. We implement experimental data from the literature to validate the model, and under various assumptions predict the dynamic behavior of p16 and other biological components by interpreting the simulation results. The quantitative model of p16-mediated pathway is created in a systematic manner in terms of Petri net technologies.

NCI60 cancer cell line panel data and RNAi analysis help identify EAF2 as a modulator of simvastatin and lovastatin response in HCT-116 cells.

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the pharmacological data available for all the NCI60 cell lines to classify simvastatin or lovastatin resistant and sensitive cell lines, respectively. Next, we performed whole-genome single marker case-control association tests for the lovastatin and simvastatin resistant and sensitive cells using their publicly available Affymetrix 125K SNP genomic data. The results were then evaluated using RNAi methodology. After correction of the p-values for multiple testing using False Discovery Rate, our results identified three genes (NRP1, COL13A1, MRPS31) and six genes (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) associated with resistance to simvastatin and lovastatin, respectively. Functional validation using RNAi confirmed that silencing of EAF2 expression modulated the response of HCT-116 colon cancer cells to both statins. In summary, we have successfully utilized the publicly available data on the NCI60 cell lines to perform whole-genome association studies for simvastatin and lovastatin. Our results indicated genes involved in the cellular response to these statins and siRNA studies confirmed the role of the EAF2 in response to these drugs in HCT-116 colon cancer cells.

Site-directed mutagenesis.

The technique of site-directed mutagenesis has been used to characterize gene and protein structure-function relationships, protein-protein interactions, binding domains of proteins, or active sites of enzymes for the last three decades. In this technique, a nucleotide sequence of interest is experimentally altered using synthetic oligonucleotides. The most commonly used approach is to use an oligonucleotide that is complementary to part of a single-stranded DNA template, but containing an internal mismatch to direct the mutation. In addition to single point mutations, this approach may also be used to construct multiple mutations, insertions, or deletions. As a result of its broad applicability in disease gene characterization studies, numerous commercial kits are now available, making this technique quick, straightforward, and reliable. In this chapter, we detail the steps involved in site-directed mutagenesis and highlight the essentials of this versatile technique based upon our experience.

RNAi-based functional pharmacogenomics.

Experimental alteration of gene expression is a powerful technique for functional characterization of disease genes. RNA interference (RNAi) is a naturally occurring mechanism of gene regulation, which is triggered by the introduction of double-stranded RNA into a cell. This phenomenon can be synthetically exploited to down-regulate expression of specific genes by transfecting mammalian cells with synthetic short interfering RNAs (siRNAs). These siRNAs can be designed to silence the expression of specific genes bearing a particular target sequence in high-throughput (HT) siRNA experimental systems and may potentially be presented as a therapeutic strategy for inhibiting transcriptional regulation of genes. This can constitute a strategy that can inhibit targets that are not tractable by small molecules such as chemical compounds. Large-scale experiments using low-dose drug exposure combined with siRNA also represent a promising discovery strategy for the purpose of identifying synergistic targets that facilitate synthetic lethal combination phenotypes. In light of such advantageous applications, siRNA technology has become an ideal research tool for studying gene function. In this chapter, we focus on the application of RNAi, with particular focus on HT siRNA phenotype profiling, to support cellular pharmacogenomics.

Genetic predisposition to ß-thalassemia and sickle cell anemia in Turkey: a molecular diagnostic approach.

The thalassemia syndromes are a diverse group of inherited disorders that can be characterized according to their insufficient synthesis or absent production of one or more of the globin chains. They are classified in to α, ß, γ, δß, δ, and ÎµÎ³Î´ß thalassemias depending on the globin chain(s) affected. The ß-thalassemias refer to that group of inherited hemoglobin disorders, which are characterized by a reduced synthesis (ß(+)-thalassemia) or absence (ß(0)-thalassemia) of beta globin (ß-globin) chain production (1). Though known as single-gene disorders, hemoglobinopathies such as ß-thalassemia and sickle cell anemia are far from being fully resolved in terms of cure, considering the less complex nature of the beta globin (ß-globin) gene family compared to more complex multifactorial genetic disorders such as cancer. Currently, there are no definitive therapeutic options for patients with ß-thalassemia and sickle cell anemia, and new insights into the pathogenesis of these devastating diseases are urgently needed. Here we address in detail the overall picture utilizing molecular diagnostic approaches that contribute to unraveling the population-specific mutational analysis of ß-globin gene. We also present approaches for molecular diagnostic strategies that are applicable to ß-thalassemia, sickle cell anemia, and other genetic disorders.

Differential subcellular expression of protein kinase C betaII in breast cancer: correlation with breast cancer subtypes.

Protein kinase C betaII (PKCßII) represents a novel potential target for anticancer therapies in breast cancer. In order to identify patient subgroups which might benefit from PKC-targeting therapies, we investigated the expression of PKCßII in human breast cancer cell lines and in a tissue microarray (TMA). We first screened breast cancer cell line representatives of breast cancer subtypes for PKCßII expression at the mRNA and at the protein levels. We analyzed a TMA comprising of tumors from 438 patients with a median followup of 15.4 years for PKCßII expression by immunohistochemistry along with other prognostic factors in breast cancer. Among a panel of human breast cancer cell lines, only MDA-MB-436, a triple negative basal cell line, showed overexpression for PKCßII both at the mRNA and at the protein levels. In breast cancer patients, cytoplasmic expression of PKCßII correlated positively with human epidermal growth factor receptor-2 (HER-2; P = 0.01) and Ki-67 (P = 0.016), while nuclear PKCßII correlated positively with estrogen receptor (ER; P = 0.016). The positive correlation of CK5/6 with cytoplasmic PKCßII (P = 0.033) lost statistical significance after adjusting for multiple comparisons (P = 0.198). Cytoplasmic PKCßII did not correlate with cyclooxygenase (COX-2; P = 0.925) and vascular endothelial growth factor (P = 1). There was no significant association between PKCßII staining and overall survival. Cytoplasmic PKCßII correlates with HER-2 and Ki-67, while nuclear PKCßII correlates with ER in breast cancer. Our study suggests the necessity for assessing the subcellular localization of PKCßII in breast cancer subtypes when evaluating the possible effectiveness of PKCßII-targeting agents.

Functional evidence implicating S100P in prostate cancer progression.

S100P protein regulates calcium signal transduction and mediates cytoskeletal interaction, protein phosphorylation and transcriptional control. We have previously shown how elevated S100P levels in prostate cancer strongly correlate with progression to metastatic disease. In our study, we evaluated the functional significance of S100P expression on prostate tumor growth in vitro and in vivo. S100P levels were modulated by overexpressing S100P in PC3 prostate cancer cells and by silencing S100P levels in 22Rv1 prostate cancer cells. Overexpression of S100P in PC3 cells promoted cell growth, increased the percentage of S-phase cells, decreased basal apoptosis rate and promoted anchorage independent growth in soft agar. Furthermore, prostate cancer cells overexpressing S100P were protected against camptothecin-induced apoptosis. Conversely, silencing of S100P in 22Rv1 cells using siRNA resulted in a prominent cytostatic effect. The influence of S100P on tumor growth and metastases were assessed in vivo. S100P-overexpressing PC3 cells had a dramatically increased tumor formation compared to controls. Microarray analysis showed the involvement of growth pathways including increased androgen receptor expression in S100P-overexpressing cells. These results provide the first functional proof that S100P overexpression can upregulate androgen receptor expression and thereby promote prostate cancer progression by increasing cell growth. Moreover, the results confirm the oncogenic nature of S100P in prostate cancer and suggest that the protein may directly confer resistance to chemotherapy. Hence, S100P could be considered a potential drug target or a chemosensitization target, and could also serve as a biomarker for aggressive, hormone-refractory and metastatic prostate cancer.

Discovery of genetic profiles impacting response to chemotherapy: application to gemcitabine.

Chemotherapy is a major treatment modality for individuals affected by cancer. Currently, a number of genome-based technologies are being adopted to identify genes associated with drug response; however, large-scale genetic association applications are still limited. Here we describe a novel strategy based on the genetic and drug response data of the NCI60 cell lines to discover potential candidate genetic variants associated with variable response to chemotherapy. As an example we have applied this strategy to discover single genetic markers and haplotypes from candidate genes previously implicated in the pharmacobiology of gemcitabine. Single-marker association analyses have implicated the association of four SNPs within the gene loci of CDC5L, EPC2, POLS, and PARP1. We have also investigated the combined effect of SNPs using haplotype-based analysis. Accordingly, we have shown modest association of haplotypes in six genes, whereas the most significant associations included a haplotype of the POLS gene. The hypothesis-generating tool presented in this study can be applied to drugs profiled in the NCI60 cell line screen and provides an effective means for the identification of genes associated with drug response. The results obtained using this novel methodology can be used to better design the clinical trials for effective study of the chemotherapeutic agents and thus provide a basis for individualized chemotherapy.

Validation of short interfering RNA knockdowns by quantitative real-time PCR.

RNA interference (RNAi) is a natural mechanism, that is triggered by the introduction of double-stranded RNA into a cell. The long double-stranded RNA is then processed into short interfering RNA (siRNA) that mediates sequence-specific degradation of homologous transcripts. This phenomenon can be exploited to experimentally trigger RNAi and downregulate gene expression by transfecting mammalian cells with synthetic siRNA. Thus, siRNAs can be designed to specifically silence the expression of genes bearing a particular target sequence. In this chapter, we present methods and procedures for validating the effects of siRNA-based gene silencing on target gene expression. To illustrate our approach, we use examples from our analysis of a Cancer Gene Library of 278 siRNAs targeting 139 classic oncogenes and tumor suppressor genes (Qiagen Inc., Germantown, MD). Specifically, this library was used for high-throughput RNAi phenotype analysis followed by gene expression analysis to validate gene silencing for siRNA that produced a phenotype. Methods and protocols are presented that illustrate how sequence-specific gene silencing of effective siRNAs are analyzed and validated by quantitative real-time PCR assays to measure the extent of target gene silencing, as well as effects on various gene expression end points.

Intersex-like (IXL) is a cell survival regulator in pancreatic cancer with 19q13 amplification.

Pancreatic cancer is a highly aggressive disease characterized by poor prognosis and vast genetic instability. Recent microarray-based, genome-wide surveys have identified multiple recurrent copy number aberrations in pancreatic cancer; however, the target genes are, for the most part, unknown. Here, we characterized the 19q13 amplicon in pancreatic cancer to identify putative new drug targets. Copy number increases at 19q13 were quantitated in 16 pancreatic cancer cell lines and 31 primary tumors by fluorescence in situ hybridization. Cell line copy number data delineated a 1.1 Mb amplicon, the presence of which was also validated in 10% of primary pancreatic tumors. Comprehensive expression analysis by quantitative real-time reverse transcription-PCR indicated that seven transcripts within this region had consistently elevated expression levels in the amplified versus nonamplified cell lines. High-throughput loss-of-function screen by RNA interference was applied across the amplicon to identify genes whose down-regulation affected cell viability. This screen revealed five genes whose down-regulation led to significantly decreased cell viability in the amplified PANC-1 cells but not in the nonamplified MiaPaca-2 cells, suggesting the presence of multiple biologically interesting genes in this region. Of these, the transcriptional regulator intersex-like (IXL) was consistently overexpressed in amplified cells and had the most dramatic effect on cell viability. IXL silencing also resulted in G(0)-G(1) cell cycle arrest and increased apoptosis in PANC-1 cells. These findings implicate IXL as a novel amplification target gene in pancreatic cancer and suggest that IXL is required for cancer cell survival in 19q13-amplified tumors.

Human SNPs resulting in premature stop codons and protein truncation.

Single nucleotide polymorphisms (SNPs) constitute the most common type of genetic variation in humans. SNPs introducing premature termination codons (PTCs), herein called X-SNPs, can alter the stability and function of transcripts and proteins and thus are considered to be biologically important. Initial studies suggested a strong selection against such variations/mutations. In this study, we undertook a genome-wide systematic screening to identify human X-SNPs using the dbSNP database. Our results demonstrated the presence of 28 X-SNPs from 28 genes with known minor allele frequencies. Eight X-SNPs (28.6 per cent) were predicted to cause transcript degradation by nonsense-mediated mRNA decay. Seventeen X-SNPs (60.7 per cent) resulted in moderate to severe truncation at the C-terminus of the proteins (deletion of >50 per cent of the amino acids). The majority of the X-SNPs (78.6 per cent) represent commonly occurring SNPs, by contrast with the rarely occurring disease-causing PTC mutations. Interestingly, X-SNPs displayed a non-uniform distribution across human populations: eight X-SNPs were reported to be prevalent across three different human populations, whereas six X-SNPs were found exclusively in one or two population(s). In conclusion, we have systematically investigated human SNPs introducing PTCs with respect to their possible biological consequences, distributions across different human populations and evolutionary aspects. We believe that the SNPs reported here are likely to affect gene/protein function, although their biological and evolutionary roles need to be further investigated.

Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer.

The identification of tumor-suppressor genes in solid tumors by classical cancer genetics methods is difficult and slow. We combined nonsense-mediated RNA decay microarrays and array-based comparative genomic hybridization for the genome-wide identification of genes with biallelic inactivation involving nonsense mutations and loss of the wild-type allele. This approach enabled us to identify previously unknown mutations in the receptor tyrosine kinase gene EPHB2. The DU 145 prostate cancer cell line, originating from a brain metastasis, carries a truncating mutation of EPHB2 and a deletion of the remaining allele. Additional frameshift, splice site, missense and nonsense mutations are present in clinical prostate cancer samples. Transfection of DU 145 cells, which lack functional EphB2, with wild-type EPHB2 suppresses clonogenic growth. Taken together with studies indicating that EphB2 may have an essential role in cell migration and maintenance of normal tissue architecture, our findings suggest that mutational inactivation of EPHB2 may be important in the progression and metastasis of prostate cancer.