Transcriptomic analysis of the adaptation to prolonged starvation of the insect-dwelling Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.

Transcriptome-Wide Analysis of microRNA-mRNA Correlations in Tissue Identifies microRNA Targeting Determinants.

MicroRNAs are small RNAs that regulate gene expression through complementary base pairing with their target mRNAs. A substantial understanding of microRNA target recognition and repression mechanisms has been reached using diverse empirical and bioinformatic approaches, primarily in vitro biochemical or cell culture perturbation settings. We sought to determine if rules of microRNA target efficacy could be inferred from extensive gene expression data of human tissues. A transcriptome-wide assessment of all the microRNA-mRNA canonical interactions' efficacy was performed using a normalized Spearman correlation (Z-score) between the abundance of the transcripts in the PRAD-TCGA dataset tissues (RNA-seq mRNAs and small RNA-seq for microRNAs, 546 samples). Using the Z-score of correlation as a surrogate marker of microRNA target efficacy, we confirmed hallmarks of microRNAs, such as repression of their targets, the hierarchy of preference for gene regions (3'UTR > CDS > 5'UTR), and seed length (6 mer < 7 mer < 8 mer), as well as the contribution of the 3'-supplementary pairing at nucleotides 13-16 of the microRNA. Interactions mediated by 6 mer + supplementary showed similar inferred repression as 7 mer sites, suggesting that the 6 mer + supplementary sites may be relevant in vivo. However, aggregated 7 mer-A1 seeds appear more repressive than 7 mer-m8 seeds, while similar when pairing possibilities at the 3'-supplementary sites. We then examined the 3'-supplementary pairing using 39 microRNAs with Z-score-inferred repressive 3'-supplementary interactions. The approach was sensitive to the offset of the bridge between seed and 3'-supplementary pairing sites, and the pattern of offset-associated repression found supports previous findings. The 39 microRNAs with effective repressive 3'supplementary sites show low GC content at positions 13-16. Our study suggests that the transcriptome-wide analysis of microRNA-mRNA correlations may uncover hints of microRNA targeting determinants. Finally, we provide a bioinformatic tool to identify microRNA-mRNA candidate interactions based on the sequence complementarity of the seed and 3'-supplementary regions.

Current Status of Regulatory Non-Coding RNAs Research in the Tritryp.

Trypanosomatids are protozoan parasites that cause devastating vector-borne human diseases. Gene expression regulation of these organisms depends on post-transcriptional control in responding to diverse environments while going through multiple developmental stages of their complex life cycles. In this scenario, non-coding RNAs (ncRNAs) are excellent candidates for a very efficient, quick, and economic strategy to regulate gene expression. The advent of high throughput RNA sequencing technologies show the presence and deregulation of small RNA fragments derived from canonical ncRNAs. This review seeks to depict the ncRNA landscape in trypanosomatids, focusing on the small RNA fragments derived from functional RNA molecules observed in RNA sequencing studies. Small RNA fragments derived from canonical ncRNAs (tsRNAs, snsRNAs, sdRNAs, and sdrRNAs) were identified in trypanosomatids. Some of these RNAs display changes in their levels associated with different environments and developmental stages, demanding further studies to determine their functional characterization and potential roles. Nevertheless, a comprehensive and detailed ncRNA annotation for most trypanosomatid genomes is still needed, allowing better and more extensive comparative and functional studies.

Hsa-miR-183-5p Modulates Cell Adhesion by Repression of ITGB1 Expression in Prostate Cancer.

Prostate cancer is a major health problem worldwide. MiR-183 is an oncomiR and a candidate biomarker in prostate cancer, affecting various pathways responsible for disease initiation and progression. We sought to discover the most relevant processes controlled by miR-183 through an unbiased transcriptomic approach using prostate cell lines and patient tissues to identify miR-183 responsive genes and pathways. Gain of function experiments, reporter gene assays, and transcript and protein measurements were conducted to validate predicted functional effects and protein mediators. A total of 135 candidate miR-183 target genes overrepresenting cell adhesion terms were inferred from the integrated transcriptomic analysis. Cell attachment, spreading assays and focal adhesion quantification of miR-183-overexpressing cells confirmed the predicted reduction in cell adhesion. ITGB1 was validated as a major target of repression by miR-183 as well as a mediator of cell adhesion in response to miR-183. The reporter gene assay and PAR-CLIP read mapping suggest that ITGB1 may be a direct target of miR-183. The negative correlation between miR-183 and ITGB1 expression in prostate cancer cohorts supports their interaction in the clinical set. Overall, cell adhesion was uncovered as a major pathway controlled by miR-183 in prostate cancer, and ITGB1 was identified as a relevant mediator of this effect.

Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves.

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication.

Extensive Translational Regulation through the Proliferative Transition of Trypanosoma cruzi Revealed by Multi-Omics.

Trypanosoma cruzi is the etiological agent for Chagas disease, a neglected parasitic disease in Latin America. Gene transcription control governs the eukaryotic cell replication but is absent in trypanosomatids; thus, it must be replaced by posttranscriptional regulatory events. We investigated the entrance into the T. cruzi replicative cycle using ribosome profiling and proteomics on G1/S epimastigote cultures synchronized with hydroxyurea. We identified 1,784 translationally regulated genes (change > 2, false-discovery rate [FDR] < 0.05) and 653 differentially expressed proteins (change > 1.5, FDR < 0.05), respectively. A major translational remodeling accompanied by an extensive proteome change is found, while the transcriptome remains largely unperturbed at the replicative entrance of the cell cycle. The differentially expressed genes comprise specific cell cycle processes, confirming previous findings while revealing candidate cell cycle regulators that undergo previously unnoticed translational regulation. Clusters of genes showing a coordinated regulation at translation and protein abundance share related biological functions such as cytoskeleton organization and mitochondrial metabolism; thus, they may represent posttranscriptional regulons. The translatome and proteome of the coregulated clusters change in both coupled and uncoupled directions, suggesting that complex cross talk between the two processes is required to achieve adequate protein levels of different regulons. This is the first simultaneous assessment of the transcriptome, translatome, and proteome of trypanosomatids, which represent a paradigm for the absence of transcriptional control. The findings suggest that gene expression chronology along the T. cruzi cell cycle is controlled mainly by translatome and proteome changes coordinated using different mechanisms for specific gene groups. IMPORTANCE Trypanosoma cruzi is an ancient eukaryotic unicellular parasite causing Chagas disease, a potentially life-threatening illness that affects 6 to 7 million people, mostly in Latin America. The antiparasitic treatments for the disease have incomplete efficacy and adverse reactions; thus, improved drugs are needed. We study the mechanisms governing the replication of the parasite, aiming to find differences with the human host, valuable for the development of parasite-specific antiproliferative drugs. Transcriptional regulation is essential for replication in most eukaryotes, but in trypanosomatids, it must be replaced by subsequent gene regulation steps since they lack transcription initiation control. We identified the genome-wide remodeling of mRNA translation and protein abundance during the entrance to the replicative phase of the cell cycle. We found that translation is strongly regulated, causing variation in protein levels of specific cell cycle processes, representing the first simultaneous study of the translatome and proteome in trypanosomatids.

Pan-cancer chromatin analysis of the human vtRNA genes uncovers their association with cancer biology.

Background: The vault RNAs (vtRNAs) are a class of 84-141-nt eukaryotic non-coding RNAs transcribed by RNA polymerase III, associated to the ribonucleoprotein complex known as vault particle. Of the four human vtRNA genes, vtRNA1-1, vtRNA1-2 and vtRNA1-3, clustered at locus 1, are integral components of the vault particle, while vtRNA2-1 is a more divergent homologue located in a second locus. Gene expression studies of vtRNAs in large cohorts have been hindered by their unsuccessful sequencing using conventional transcriptomic approaches. Methods: VtRNA expression in The Cancer Genome Atlas (TCGA) Pan-Cancer cohort was estimated using the genome-wide DNA methylation and chromatin accessibility data (ATAC-seq) of their genes as surrogate variables. The association between vtRNA expression and patient clinical outcome, immune subtypes and transcriptionally co-regulated gene programs was analyzed in the dataset. Results: VtRNAs promoters are enriched in transcription factors related to viral infection. VtRNA2-1 is likely the most independently regulated homologue. VtRNA1-1 has the most accessible chromatin, followed by vtRNA1-2, vtRNA2-1 and vtRNA1-3. VtRNA1-1 and vtRNA1-3 chromatin status does not significantly change in cancer tissues. Meanwhile, vtRNA2-1 and vtRNA1-2 expression is widely deregulated in neoplastic tissues and its alteration is compatible with a broad oncogenic role for vtRNA1-2, and both tumor suppressor and oncogenic functions for vtRNA2-1. Yet, vtRNA1-1, vtRNA1-2 and vtRNA2-1 promoter DNA methylation predicts a shorter patient overall survival cancer-wide. In addition, gene ontology analyses of vtRNAs co-regulated genes identify a chromosome regulatory domain, epithelial differentiation, immune and thyroid cancer gene sets for specific vtRNAs. Furthermore, vtRNA expression patterns are associated with cancer immune subtypes and vtRNA1-2 expression is positively associated with cell proliferation and wound healing. Conclusions: Our study presents the landscape of vtRNA chromatin status cancer-wide, identifying co-regulated gene networks and ontological pathways associated with the different vtRNA genes that may account for their diverse roles in cancer.

vtRNA2-1/nc886 Produces a Small RNA That Contributes to Its Tumor Suppression Action through the microRNA Pathway in Prostate Cancer.

vtRNA2-1 is a vault RNA initially classified as microRNA precursor hsa-mir-886 and recently proposed as "nc886", a new type of non-coding RNA involved in cancer progression acting as an oncogene and tumor suppressor gene in different tissues. We have shown that vtRNA2-1/nc886 is epigenetically repressed in neoplastic cells, increasing cell proliferation and invasion in prostate tissue. Here we investigate the ability of vtRNA2-1/nc886 to produce small-RNAs and their biological effect in prostate cells. The interrogation of public small-RNA transcriptomes of prostate and other tissues uncovered two small RNAs, snc886-3p and snc886-5p, derived from vtRNA2-1/nc886 (previously hsa-miR-886-3p and hsa-miR-886-5p). Re-analysis of PAR-CLIP and knockout of microRNA biogenesis enzymes data showed that these small RNAs are products of DICER, independent of DROSHA, and associate with Argonaute proteins, satisfying microRNA attributes. In addition, the overexpression of snc886-3p provokes the downregulation of mRNAs bearing sequences complementary to its "seed" in their 3'-UTRs. Microarray and in vitro functional assays in DU145, LNCaP and PC3 cell lines revealed that snc886-3p reduced cell cycle progression and increases apoptosis, like its precursor vtRNA2-1/nc886. Finally, we found a list of direct candidate targets genes of snc886-3p upregulated and associated with disease condition and progression in PRAD-TCGA data. Overall, our findings suggest that vtRNA2-1/nc886 and its processed product snc886-3p are synthesized in prostate cells, exerting a tumor suppressor action.

Isolation and Structural Characterization of Bioactive Molecules on Prostate Cancer from Mayan Traditional Medicinal Plants.

Prostate cancer is the most common cancer in men around the world. It is a complex and heterogeneous disease in which androgens and their receptors play a crucial role in the progression and development. The current treatment for prostate cancer is a combination of surgery, hormone therapy, radiation and chemotherapy. Therapeutic agents commonly used in the clinic include steroidal and non-steroidal anti-androgens, such as cyproterone acetate, bicalutamide and enzalutamide. These few agents have multiple adverse effects and are not 100% effective. Several plant compounds and mixtures, including grape seed polyphenol extracts, lycopene and tomato preparations, soy isoflavones, and green tea extracts, have been shown to be effective against prostate cancer cell growth. In vivo activity of some isolated compounds like capsaicin and curcumin was reported in prostate cancer murine models. We prepared a library of plant extracts from traditional Mayan medicine. These plants were selected for their use in the contemporaneous Mayan communities for the treatment of different diseases. The extracts were assessed in a phenotypic screening using LNCaP prostate cancer androgen sensitive cell line, with a fixed dose of 25 µg/mL. MTT assay identified seven out of ten plants with interesting anti-neoplastic activity. Extracts from these plants were subjected to a bioguided fractionation to study their major components. We identified three compounds with anti-neoplastic effects against LNCaP cells, one of which shows selectivity for neoplastic compared to benign cells.

An integrated view of the role of miR-130b/301b miRNA cluster in prostate cancer.

Prostate cancer is a major health problem worldwide due to its high incidence morbidity and mortality. There is currently a need of improved biomarkers, capable to distinguish mild versus aggressive forms of the disease, and thus guide therapeutic decisions. Although miRNAs deregulated in cancer represent exciting candidates as biomarkers, its scientific literature is frequently fragmented in dispersed studies. This problem is aggravated for miRNAs belonging to miRNA gene clusters with shared target genes. The miRNA cluster composed by hsa-mir-130b and hsa-mir-301b precursors was recently involved in prostate cancer pathogenesis, yet different studies assigned it opposite effects on the disease. We sought to elucidate the role of the human miR-130b/301b miRNA cluster in prostate cancer through a comprehensive data analysis of most published clinical cohorts. We interrogated methylomes, transcriptomes and patient clinical data, unifying previous reports and adding original analysis using the largest available cohort (TCGA-PRAD). We found that hsa-miR-130b-3p and hsa-miR-301b-3p are upregulated in neoplastic vs normal prostate tissue, as well as in metastatic vs primary sites. However, this increase in expression is not due to a decrease of the global DNA methylation of the genes in prostate tissues, as the promoter of the gene remains lowly methylated in normal and neoplastic tissue. A comparison of the levels of human miR-130b/301b and all the clinical variables reported for the major available cohorts, yielded positive correlations with malignance, specifically significant for T-stage, residual tumor status and primary therapy outcome. The assessment of the correlations between the hsa-miR-130b-3p and hsa-miR-301b-3p and candidate target genes in clinical samples, supports their repression of tumor suppressor genes in prostate cancer. Altogether, these results favor an oncogenic role of miR-130b/301b cluster in prostate cancer.

Nc886 is epigenetically repressed in prostate cancer and acts as a tumor suppressor through the inhibition of cell growth.

BACKGROUND: Nc886 is a 102 bp non-coding RNA transcript initially classified as a microRNA precursor (Pre-miR-886), later as a divergent homologue of the vault RNAs (vtRNA 2-1) and more recently as a novel type of RNA (nc886). Although nc886/vtRNA2-1/Pre-miR-886 identity is still controversial, it was shown to be epigenetically controlled, presenting both tumor suppressor and oncogenic function in different cancers. Here, we study for the first time the role of nc886 in prostate cancer. METHODS: Nc886 promoter methylation status and its correlation with patient clinical parameters or DNMTs levels were evaluated in TCGA and specific GEO prostate tissue datasets. Nc886 level was measured by RT-qPCR to compare normal/neoplastic prostate cells from radical prostatectomies and cell lines, and to assess nc886 response to demethylating agents. The effect of nc886 recovery in cell proliferation (in vitro and in vivo) and invasion (in vitro) was evaluated using lentiviral transduced DU145 and LNCaP cell lines. The association between the expression of nc886 and selected genes was analyzed in the TCGA-PRAD cohort. RESULTS: Nc886 promoter methylation increases in tumor vs. normal prostate tissue, as well as in metastatic vs. normal prostate tissue. Additionally, nc886 promoter methylation correlates with prostate cancer clinical staging, including biochemical recurrence, Clinical T-value and Gleason score. Nc886 transcript is downregulated in tumor vs. normal tissue -in agreement with its promoter methylation status- and increases upon demethylating treatment. In functional studies, the overexpression of nc886 in the LNCaP and DU145 cell line leads to a decreased in vitro cell proliferation and invasion, as well as a reduced in vivo cell growth in NUDE-mice tumor xenografts. Finally, nc886 expression associates with the prostate cancer cell cycle progression gene signature in TCGA-PRAD. CONCLUSIONS: Our data suggest a tumor suppressor role for nc886 in the prostate, whose expression is epigenetically silenced in cancer leading to an increase in cell proliferation and invasion. Nc886 might hold clinical value in prostate cancer due to its association with clinical parameters and with a clinically validated gene signature.

Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control.

Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.

BACKGROUND: Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms. RESULTS: Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect's life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage. CONCLUSIONS: A large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.

Conserved Curvature of RNA Polymerase I Core Promoter Beyond rRNA Genes: The Case of the Tritryps.

In trypanosomatids, the RNA polymerase I (RNAPI)-dependent promoters controlling the ribosomal RNA (rRNA) genes have been well identified. Although the RNAPI transcription machinery recognizes the DNA conformation instead of the DNA sequence of promoters, no conformational study has been reported for these promoters. Here we present the in silico analysis of the intrinsic DNA curvature of the rRNA gene core promoters in Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. We found that, in spite of the absence of sequence conservation, these promoters hold conformational properties similar to other eukaryotic rRNA promoters. Our results also indicated that the intrinsic DNA curvature pattern is conserved within the Leishmania genus and also among strains of T. cruzi and T. brucei. Furthermore, we analyzed the impact of point mutations on the intrinsic curvature and their impact on the promoter activity. Furthermore, we found that the core promoters of protein-coding genes transcribed by RNAPI in T. brucei show the same conserved conformational characteristics. Overall, our results indicate that DNA intrinsic curvature of the rRNA gene core promoters is conserved in these ancient eukaryotes and such conserved curvature might be a requirement of RNAPI machinery for transcription of not only rRNA genes but also protein-coding genes.

Implication of CA repeated tracts on post-transcriptional regulation in Trypanosoma cruzi.

In Trypanosoma cruzi gene expression regulation mainly relays on post-transcriptional events. Nevertheless, little is known about the signals which control mRNA abundance and functionality. We have previously found that CA repeated tracts (polyCA) are abundant in the vicinity of open reading frames and constitute specific targets for single stranded binding proteins from T. cruzi epimastigote. Given the reported examples of the involvement of polyCA motifs in gene expression regulation, we decided to further study their role in T. cruzi. Using an in silico genome-wide analysis, we identify the genes that contain polyCA within their predicted UTRs. We found that about 10% of T. cruzi genes carry polyCA therein. Strikingly, they are frequently concurrent with GT repeated tracts (polyGT), favoring the formation of a secondary structure exhibiting the complementary polydinucleotides in a double stranded helix. This feature is found in the species-specific family of genes coding for mucine associated proteins (MASPs) and other genes. For those polyCA-containing UTRs that lack polyGT, the polyCA is mainly predicted to adopt a single stranded structure. We further analyzed the functional role of such element using a reporter approach in T. cruzi. We found out that the insertion of polyCA at the 3' UTR of a reporter gene in the pTEX vector modulates its expression along the parasite's life cycle. While no significant change of the mRNA steady state of the reporter gene could be detected at the trypomastigote stage, significant increase in the epimastigote and reduction in the amastigote stage were observed. Altogether, these results suggest the involvement of polyCA as a signal in gene expression regulation in T. cruzi.

Comparative genomic analysis of dinucleotide repeats in Tritryps.

The protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryps), are evolutionarily ancient eukaryotes which cause worldwide human parasitosis. They present unique biological features. Indeed, canonical DNA/RNA cis-acting elements remain mostly elusive. Repetitive sequences, originally considered as selfish DNA, have been lately recognized as potentially important functional sequence elements in cell biology. In particular, the dinucleotide patterns have been related to genome compartmentalization, gene evolution and gene expression regulation. Thus, we perform a comparative analysis of the occurrence, length and location of dinucleotide repeats (DRs) in the Tritryp genomes and their putative associations with known biological processes. We observe that most types of DRs are more abundant than would be expected by chance. Complementary DRs usually display asymmetrical strand distribution, favoring TT and GT repeats in the coding strands. In addition, we find that GT repeats are among the longest DRs in the three genomes. We also show that specific DRs are non-uniformly distributed along the polycistronic unit, decreasing toward its boundaries. Distinctive non-uniform density patterns were also found in the intergenic regions, with predominance at the vicinity of the ORFs. These findings further support that DRs may control genome structure and gene expression.

Pharmacogenomics and cancer stem cells: a changing landscape?

Pharmacogenomics in oncology holds the promise to personalize cancer therapy. However, its clinical application is still limited to a few genes, and, in the large majority of cancers, the correlation between genotype and clinical outcome has been disappointing. One possible explanation is that current pharmacogenomic studies do not take into account the emerging role of cancer stem cells (CSCs) in drug sensitivity and resistance. CSCs are a subpopulation of cells driven by specific signal-transduction pathways, but genetic variants affecting their activity are generally neglected in current pharmacogenomic studies. Moreover, in several malignancies, CSCs represent a rare sub-population; therefore, whole tumor profiling might mask CSC gene expression patterns. This article reviews current evidence on CSC chemoresistance and shows how common genetic variations in CSC-related genes may predict individual response to anti-cancer agents. Furthermore, we provide insights into the design of pharmacogenomic studies to address the clinical usefulness of CSC genetic profiling.

Genomic profiling of tumor initiating prostatospheres.

BACKGROUND: The cancer stem cell (CSC) hypothesis proposes that a population of tumor cells bearing stem cell properties is responsible for the origin and maintenance of tumors. Normal and cancer stem cells possess the ability to grow in vitro as self-renewing spheres, but the molecular basis of this phenotype remains largely unknown. We intended to establish a comprehensive culture system to grow prostatospheres (PSs) from both cancer cell lines and patient tumors. We then used gene expression microarrays to gain insight on the molecular pathways that sustain the PS tumor initiating cell (TIC) phenotype. RESULTS: Traditional stem cell medium (SCM) supplemented with KnockoutSR (KO) allows the propagation of monoclonal PSs from cell lines and primary cells. PSs display gene expression and tumorigenicity hallmarks of TICs. Gene expression analysis defined a gene signature composed of 66 genes that characterize LNCaP and patient PSs. This set includes novel prostate TIC growth factors (NRP1, GDF1, JAG1), proteins implicated in cell adhesion and cytoskeletal maintenance, transcriptional regulators (MYCBP, MYBL1, ID1, ID3, FOS, ELF3, ELF4, KLF2, KLF5) and factors involved in protein biosynthesis and metabolism. Meta-analysis in Oncomine reveals that some of these genes correlate with prostate cancer status and/or progression. Reporter genes and inhibitors indicate that the Notch pathway contributes to prostatosphere growth. CONCLUSIONS: We have developed a model for the culture of PSs, and provide a genomic profile that support CSCs identity. This signature identifies novel markers and pathways that are predicted to correlate with prostate cancer evolution.

Long-range enhancers on 8q24 regulate c-Myc.

Recent genomewide association studies have found multiple genetic variants on chromosome 8q24 that are significantly associated with an increased susceptibility to prostate, colorectal, and breast cancer. These risk loci are located in a "gene desert," a few hundred kilobases telomeric to the Myc gene. To date, the biological mechanism(s) underlying these associations remain unclear. It has been speculated that these 8q24 genetic variant(s) might affect Myc expression by altering its regulation or amplification status. Here, we show that multiple enhancer elements are present within this region and that they can regulate transcription of Myc. We also demonstrate that one such enhancer element physically interacts with the Myc promoter via transcription factor Tcf-4 binding and acts in an allele specific manner to regulate Myc expression.

Effects of the sesquiterpene lactone parthenolide on prostate tumor-initiating cells: An integrated molecular profiling approach.

Recent evidence suggests tumor-initating cells (TICs), also called cancer stem cells, are responsible for tumor initiation and progression; therefore, they represent an important cell population for development of future anti-cancer therapies. In this study, we show that the sesquiterpene lactone parthenolide (PTL) is cytotoxic to prostate TICs isolated from prostate cancer cell lines: DU145, PC3, VCAP, and LAPC4, as well as primary prostate TICs. Furthermore, PTL inhibited TIC-driven tumor formation in mouse xenografts. Using an integrated molecular profiling approach encompassing proteomics, profiles of activated transcription factors and genomics we ascertained the effects of PTL on prostate cancer cells. In addition to the previously described effects of PTL, we determined that the non-receptor tyrosine kinase src, and many src signaling components, including: Csk, FAK, beta1-arrestin, FGFR2, PKC, MEK/MAPK, CaMK, ELK-1, and ELK-1-dependent genes are novel targets of PTL action. Furthermore, PTL altered the binding of transcription factors important in prostate cancer including: C/EBP-alpha, fos related antigen-1 (FRA-1), HOXA-4, c-MYB, SNAIL, SP1, serum response factor (SRF), STAT3, X-box binding protein-1 (XBP1), and p53. In summary, we show PTL is cytotoxic to prostate TICs and describe the molecular events of PTL-mediated cytotoxicity. Therefore, PTL represents a promising therapeutic for prostate cancer treatment.