Genomic profiling of tumor initiating prostatospheres

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Traditional stem cell medium (SCM) supplemented with Knockout™SR (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.</p> <p>Conclusions</p> <p>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.</p

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

STAT3 inhibition with Galiellalactone effectively targets the prostate cancer stem-like cell population."

Cancer stem cells (CSCs) are a small subpopulation of quiescent cells with the potential to differentiate into tumor cells. CSCs are involved in tumor initiation and progression and contribute to treatment failure through their intrinsic resistance to chemo- or radiotherapy, thus representing a substantial concern for cancer treatment. Prostate CSCs’ activity has been shown to be regulated by the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Here we investigated the effect of galiellalactone (GL), a direct STAT3 inhibitor, on CSCs derived from prostate cancer patients, on docetaxel-resistant spheres with stem cell characteristics, on CSCs obtained from the DU145 cell line in vitro and on DU145 tumors in vivo. We found that GL significantly reduced the viability of docetaxel-resistant and patient-derived spheres. Moreover, CSCs isolated from DU145 cells were sensitive to low concentrations of GL, and the treatment with GL suppressed their viability and their ability to form colonies and spheres. STAT3 inhibition down regulated transcriptional targets of STAT3 in these cells, indicating STAT3 activity in CSCs. Our results indicate that GL can target the prostate stem cell niche in patient-derived cells, in docetaxel-resistant spheres and in an in vitro model. We conclude that GL represents a promising therapeutic approach for prostate cancer patients, as it reduces the viability of prostate cancer-therapy-resistant cells in both CSCs and non-CSC populations

Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

<p>Abstract</p> <p>Background</p> <p>Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I).</p> <p>Methods</p> <p>Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and <it>in vitro </it>radioresistance assay were carried out under standard conditions.</p> <p>Results</p> <p>CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 10³dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 10⁵monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR).</p> <p>Conclusions</p> <p>We characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, SiHa, Ca Ski and C-4 I) and found that they express characteristic markers of stem cell, EMT and radioresistance. The fact that CICs demonstrated a higher degree of resistance to radiation than differentiated cells suggests that specific detection and targeting of CICs could be highly valuable for the therapy of tumors from the uterine cervix.</p

Pathobiological Implications of the Expression of EGFR, pAkt, NF-κB and MIC-1 in Prostate Cancer Stem Cells and Their Progenies

The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse. The present study was undertaken to establish the possibility of using a combination of specific oncogenic products, including epidermal growth factor receptor (EGFR), pAkt, nuclear factor-kappaB (NF-κB) and macrophage inhibitory cytokine-1 (MIC-1) as biomarkers and therapeutic targets for optimizing the management of patients with localized PC at earlier disease stages. The immunohistochemical and immunofluorescence data have revealed that the expression levels of EGFR, Ser473-pAkt, NF-κB p65 and MIC-1 proteins were significantly enhanced in the same subset of 76 cases of prostatic adenocarcinoma specimens during the disease progression and these biomarkers were expressed in a small subpopulation of CD133+ PC cells and the bulk tumor mass of CD133− PC cells. Importantly, all of these biomarkers were also overexpressed in 80–100% of 30 PC metastasis bone tissue specimens. Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells. Of therapeutic interest, the targeting of EGFR, pAkt, NF-κB or MIC-1 was also effective at suppressing the basal and EGF-promoted prostasphere formation by SP WPE1-NB26 cells, inducing disintegration of SP cell-derived prostaspheres and decreasing the viability of SP and non-SP WPE1-NB26 cell fractions. Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel. These findings suggest that the combined use of EGFR, pAkt, NF-κB and/or MIC-1 may represent promising strategies for improving the accuracy of current diagnostic and prognostic methods and efficacy of treatments of PC patients in considering the disease heterogeneity, thereby preventing PC progression to metastatic and lethal disease states

DNA repair: the culprit for tumor-initiating cell survival?

The existence of “tumor-initiating cells” (TICs) has been a topic of heated debate for the last few years within the field of cancer biology. Their continuous characterization in a variety of solid tumors has led to an abundance of evidence supporting their existence. TICs are believed to be responsible for resistance against conventional treatment regimes of chemotherapy and radiation, ultimately leading to metastasis and patient demise. This review summarizes DNA repair mechanism(s) and their role in the maintenance and regulation of stem cells. There is evidence supporting the hypothesis that TICs, similar to embryonic stem (ES) cells and hematopoietic stem cells (HSCs), display an increase in their ability to survive genotoxic stress and injury. Mechanistically, the ability of ES cells, HSCs and TICs to survive under stressful conditions can be attributed to an increase in the efficiency at which these cells undergo DNA repair. Furthermore, the data presented in this review summarize the results found by our lab and others demonstrating that TICs have an increase in their genomic stability, which can allow for TIC survival under conditions such as anticancer treatments, while the bulk population of tumor cells dies. We believe that these data will greatly impact the development and design of future therapies being engineered to target and eradicate this highly aggressive cancer cell population

Silencing of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer

The Kruppel-like factor (KLF) proteins are multitasked transcriptional regulators with an expanding tumor suppressor function. KLF2 is one of the prominent members of the family because of its diminished expression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic roles. In this study, we show that epigenetic silencing of KLF2 occurs in cancer cells through direct transcriptional repression mediated by the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2). Binding of EZH2 to the 5′-end of KLF2 is also associated with a gain of trimethylated lysine 27 histone H3 and a depletion of phosphorylated serine 2 of RNA polymerase. Upon depletion of EZH2 by RNA interference, short hairpin RNA or use of the small molecule 3-Deazaneplanocin A, the expression of KLF2 was restored. The transfection of KLF2 in cells with EZH2-associated silencing showed a significant anti-tumoral effect, both in culture and in xenografted nude mice. In this last setting, KLF2 transfection was also associated with decreased dissemination and lower mortality rate. In EZH2-depleted cells, which characteristically have lower tumorigenicity, the induction of KLF2 depletion ‘rescued' partially the oncogenic phenotype, suggesting that KLF2 repression has an important role in EZH2 oncogenesis. Most importantly, the translation of the described results to human primary samples demonstrated that patients with prostate or breast tumors with low levels of KLF2 and high expression of EZH2 had a shorter overall survival

Breakdown of phylogenetic signal: a survey of microsatellite densities in 454 shotgun sequences from 154 non model Eukaryote species

Microsatellites are ubiquitous in Eukaryotic genomes. A more complete understanding of their origin and spread can be gained from a comparison of their distribution within a phylogenetic context. Although information for model species is accumulating rapidly, it is insufficient due to a lack of species depth, thus intragroup variation is necessarily ignored. As such, apparent differences between groups may be overinflated and generalizations cannot be inferred until an analysis of the variation that exists within groups has been conducted. In this study, we examined microsatellite coverage and motif patterns from 454 shotgun sequences of 154 Eukaryote species from eight distantly related phyla (Cnidaria, Arthropoda, Onychophora, Bryozoa, Mollusca, Echinodermata, Chordata and Streptophyta) to test if a consistent phylogenetic pattern emerges from the microsatellite composition of these species. It is clear from our results that data from model species provide incomplete information regarding the existing microsatellite variability within the Eukaryotes. A very strong heterogeneity of microsatellite composition was found within most phyla, classes and even orders. Autocorrelation analyses indicated that while microsatellite contents of species within clades more recent than 200 Mya tend to be similar, the autocorrelation breaks down and becomes negative or non-significant with increasing divergence time. Therefore, the age of the taxon seems to be a primary factor in degrading the phylogenetic pattern present among related groups. The most recent classes or orders of Chordates still retain the pattern of their common ancestor. However, within older groups, such as classes of Arthropods, the phylogenetic pattern has been scrambled by the long independent evolution of the lineages.Emese Meglécz, Gabriel Nève, Ed Biffin and Michael G. Gardne

Identification of Vitronectin as an Extrinsic Inducer of Cancer Stem Cell Differentiation and Tumor Formation

There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin alpha V beta 3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of beta-catenin nuclear localization. Blocking integrin alpha V beta 3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways