An XIST-related small RNA regulates KRAS G-quadruplex formation beyond X-inactivation.

X-inactive-specific transcript (XIST), a long non-coding RNA, is essential for the initiation of X-chromosome inactivation. However, little is known about other roles of XIST in the physiological process in eukaryotic cells. In this study, the bioinformatics approaches revealed XIST could be processed into a small non-coding RNA XPi2. The XPi2 RNA was confirmed by a northern blot assay; its expression was gender-independent, suggesting the role of XPi2 was beyond X-chromosome inactivation. The pull-down assay combined with LC-MS-MS identified two XPi2-associated proteins, nucleolin and hnRNP A1, connected to the formation of G-quadruplex. Moreover, the microarray data showed the knockdown of XPi2 down-regulated the KRAS pathway. Consistently, we tested the expression of ten genes, including KRAS, which was correlated with a G-quadruplex formation and found the knockdown of XPi2 caused a dramatic decrease in the transcription level of KRAS among the ten genes. The results of CD/NMR assay also supported the interaction of XPi2 and the polypurine-polypyrimidine element of KRAS. Accordingly, XPi2 may stimulate the KRAS expression by attenuating G-quadruplex formation. Our present work sheds light on the novel role of small RNA XPi2 in modulating the G-quadruplex formation which may play some essential roles in the KRAS- associated carcinogenesis.

Therapeutic potential of microRNA let-7: tumor suppression or impeding normal stemness.

The first microRNA, let-7, and its family were discovered in Caenorhabditis elegans and are functionally conserved from worms to humans in the regulation of embryonic development and stemness. The let-7 family has been shown to have an essential role in stem cell differentiation and tumor-suppressive activity. Deregulating expression of let-7 is commonly reported in many human cancers. Emerging evidence has accumulated and suggests that reestablishment of let-7 in tumor cells is a valuable therapeutic strategy. However, findings reach beyond tumor therapeutics and may impinge on stemness and differentiation of stem cells. In this review, we discuss the role of let-7 in development and differentiation of normal adult stem/progenitor cells and offer a viewpoint of the association between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the application of let-7 are highlighted.

Targeting cancer stem cells for treatment of glioblastoma multiforme.

Cancer stem cells (CSCs) in glioblastoma multiforme (GBM) are radioresistant and chemoresistant, which eventually results in tumor recurrence. Targeting CSCs for treatment is the most crucial issue. There are five methods for targeting the CSCs of GBM. One is to develop a new chemotherapeutic agent specific to CSCs. A second is to use a radiosensitizer to enhance the radiotherapy effect on CSCs. A third is to use immune cells to attack the CSCs. In a fourth method, an agent is used to promote CSCs to differentiate into normal cells. Finally, ongoing gene therapy may be helpful. New therapeutic agents for targeting a signal pathway, such as epidermal growth factor (EGF) and vascular epidermal growth factor (VEGF) or protein kinase inhibitors, have been used for GBM but for CSCs the effects still require further evaluation. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as cyclooxygenase-2 (Cox-2) inhibitors have proven to be effective for increasing radiation sensitivity of CSCs in culture. Autologous dendritic cells (DCs) are one of the promising immunotherapeutic agents in clinical trials and may provide another innovative method for eradication of CSCs. Bone-morphogenetic protein 4 (BMP4) is an agent used to induce CSCs to differentiate into normal glial cells. Research on gene therapy by viral vector is also being carried out in clinical trials. Targeting CSCs by eliminating the GBM tumor may provide an innovative way to reduce tumor recurrence by providing a synergistic effect with conventional treatment. The combination of conventional surgery, chemotherapy, and radiotherapy with stem cell-orientated therapy may provide a new promising treatment for reducing GBM recurrence and improving the survival rate.

Transcribed pseudogene ψPPM1K generates endogenous siRNA to suppress oncogenic cell growth in hepatocellular carcinoma.

Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene ψPPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted ψPPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected ψPPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of ψPPM1K. Furthermore, expression of NEK8 in ψPPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of ψPPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth.

pseudoMap: an innovative and comprehensive resource for identification of siRNA-mediated mechanisms in human transcribed pseudogenes.

RNA interference (RNAi) is a gene silencing process within living cells, which is controlled by the RNA-induced silencing complex with a sequence-specific manner. In flies and mice, the pseudogene transcripts can be processed into short interfering RNAs (siRNAs) that regulate protein-coding genes through the RNAi pathway. Following these findings, we construct an innovative and comprehensive database to elucidate siRNA-mediated mechanism in human transcribed pseudogenes (TPGs). To investigate TPG producing siRNAs that regulate protein-coding genes, we mapped the TPGs to small RNAs (sRNAs) that were supported by publicly deep sequencing data from various sRNA libraries and constructed the TPG-derived siRNA-target interactions. In addition, we also presented that TPGs can act as a target for miRNAs that actually regulate the parental gene. To enable the systematic compilation and updating of these results and additional information, we have developed a database, pseudoMap, capturing various types of information, including sequence data, TPG and cognate annotation, deep sequencing data, RNA-folding structure, gene expression profiles, miRNA annotation and target prediction. As our knowledge, pseudoMap is the first database to demonstrate two mechanisms of human TPGs: encoding siRNAs and decoying miRNAs that target the parental gene. pseudoMap is freely accessible at http://pseudomap.mbc.nctu.edu.tw/. Database URL: http://pseudomap.mbc.nctu.edu.tw/

Adjuvant immunotherapy with whole-cell lysate dendritic cells vaccine for glioblastoma multiforme: a phase II clinical trial.

BACKGROUND: This study sought to evaluate effectiveness of autologous dendritic cell vaccine (immunotherapy) for glioblastoma multiforme (GBM). METHODS: Patients 14 to 70 years of age with newly diagnosed GBM and Karnofsky Performance Scale (KPS) score >70 who were receiving initial treatment were enrolled and were randomized into 2 groups during the 5-year study period. Eighteen patients underwent conventional treatment (surgery, radiotherapy, and chemotherapy) and received adjuvant autologous dendritic cell vaccine, and 16 patients (control group) underwent conventional treatment only. Administration of the vaccine was begun within 1 to 2 months postoperatively, with 10 inoculations given over 6 months. Outcome measures were overall survival (OS); progression-free survival (PFS); 1-, 2-, and 3-year survival rates, and quality of life (QoL). RESULTS: Follow-up time ranged from 14 to 56 months (median, 33 months). The 1-, 2-, and 3-year survival rates were 88.9%, 44.4%, and 16.7% for the vaccine group, respectively, and 75.0%, 18.8%, and 0%, respectively, for the control group, (P = 0.299, 0.0035, 0.0014, respectively). The median OS for the vaccine group was 31.9 months and for the control group was 15.0 months (P < 0.002). The median progression-free survival (PFS) for the vaccine group was 8.5 months, and 8.0 months for the control group (P = 0.075). The surviving fraction was significantly higher in the vaccine group based on Kaplan-Meier analysis. CONCLUSIONS: Adjuvant immunotherapy with whole-cell lysate dendritic cell vaccine may improve short-term survival. It seems to be safe, and its long-term effectiveness is worthy of further investigation.

A phase I/II clinical trial investigating the adverse and therapeutic effects of a postoperative autologous dendritic cell tumor vaccine in patients with malignant glioma.

Previous clinical trials of dendritic cell (DC)-based immunotherapy in patients with glioblastoma multiforme (GBM) have reported induction of systemic immune responses and prolonged survival. From 2003 to 2005, we performed a clinical trial in which patients with malignant glioma underwent surgery for maximal cytoreduction followed by a 6-month 10-injection course of autologous DC-tumor vaccine therapy, each injection containing 1-6×10(7) DC. Of the 17 treated patients (16 with World Health Organization grade IV and one with grade III glioma), eight (47.1%) had an initial transient elevation in aspartate aminotransferase (AST)/alanine aminotransferase (ALT). Vaccination caused some tumor shrinkage and increased concentration of tumor-infiltrating CD8(+) lymphocytes. Median survival and 5-year survival were 525 days and 18.8%, respectively, for 16 patients with grade IV glioma (381 days and 12.5% for eight newly diagnosed; 966 days and 25% for eight relapsed patients) compared to 380 days and 0% for 63 historical control patients. We concluded that autologous DC-tumor immunotherapy benefits patients with malignant glioma but may cause transient but reversible elevation of serum AST/ALT levels.

Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.

Amiloride modulates alternative splicing in leukemic cells and resensitizes Bcr-AblT315I mutant cells to imatinib.

The antihypertensive drug amiloride is being considered as a tactic to improve cancer therapy including that for chronic myelogenous leukemia. In this study, we show that amiloride modulates the alternative splicing of various cancer genes, including Bcl-x, HIPK3, and BCR/ABL, and that this effect is not mainly related to pH alteration, which is a known effect of the drug. Splice modulation involved various splicing factors, with the phosphorylation state of serine-arginine-rich (SR) proteins also altered during the splicing process. Pretreatment with okadaic acid to inhibit protein phosphatase PP1 reversed partially the phosphorylation levels of SR proteins and also the amiloride-modulated yields of Bcl-xs and HIPK3 U(-) isoforms. Genome-wide detection of alternative splicing further revealed that many other apoptotic genes were regulated by amiloride, including APAF-1, CRK, and SURVIVIN. Various proteins of the Bcl-2 family and MAPK kinases were found to be involved in amiloride-induced apoptosis. Moreover, the effect of amiloride on mRNA levels of Bcl-x was demonstrated to translate to the protein levels. Cotreatment of K562 and BaF3/Bcr-AblT315I cells with amiloride and imatinib induced more loss of cell viability than either agent alone. Our findings suggest that amiloride may offer a potential treatment option for chronic myelogenous leukemia either alone or in combination with imatinib.

The role of cancer stem cells (CD133(+)) in malignant gliomas.

Malignant gliomas, particularly glioblastoma multiforme (GBM) tumors, are very difficult to treat by conventional approaches. Although most of the tumor mass can be removed by surgical resection, radiotherapy, and chemotherapy, it eventually recurs. There is growing evidence that cancer stem cells (CSCs) play an important role in tumor recurrence. These stem cells are radioresistant and chemoresistant. The most commonly used tumor marker for CSCs is CD133. The amount of CSC component is closely correlated with tumor malignancy grading. Isolating, identifying, and treating CSCs as the target is crucial for treating malignant gliomas. CSC-associated vascular endothelial growth factor (VEGF) promotes tumor angiogenesis, tumor hemorrhage, and tumor infiltration. Micro-RNA (miRNA) plays a role in CSC gene expression, which may regulate oncogenesis or suppression to influence tumor development or progression. The antigenesis of CSCs and normal stem cells may be different. The CSCs may escape the T-cell immune response. Identifying a new specific antigen from CSCs for vaccine treatment is a key point for immunotherapy. On the other hand, augmented treatment with radiosensitizer or chemosensitizer may lead to reduction of CSCs and lead to CSCs being vulnerable to radiotherapy and chemotherapy. The control of signaling pathway and cell differentiation to CSC growth is another new hope for treatment of malignant gliomas. Although the many physiological behavioral differences between CSCs and normal stem cells are unclear, the more we know about these differences the better we will be able to treat CSCs effectively.

Recent advances of dendritic cells (DCs)-based immunotherapy for malignant gliomas.

Immunotherapy is a new light of hope for the treatment of malignant gliomas. The brain is no longer believed to be an immunologically privileged organ. The major advantage of immunotherapy is the tumor-specific cytotoxic effect on the tumor cells with minimal side effects. Autologous dendritic cells (DCs)-based immunotherapy is a promising and feasible method. DCs are the most potent antigen-presenting cells (APCs). DCs prime T lymphocytes by epitopic major histocompatibility (MHC) class I and II for CD8(+) cytotoxic T lymphocytes (CTLs) and CD4(+) T helper cells, respectively. From the tissue specimen examination after DCs-based immunotherapy, CD8(+) CTLs have replaced T regulatory cells (Tregs) as the major dominant tissue infiltrating lymphocytes (TILs). CD8(+) CTLs play a key role in the tumor response, which may also be effective against cancer stem cells. DCs themselves also produce many cytokines including interferon-gamma and interleukin (IL-2) to kill the tumor cells. From the preliminary better outcomes in the literature for malignant gliomas, DC-based immunotherapy may improve tumor response by increasing the survival rate and time. It is recommended that DC-based immunotherapy is applied as soon as possible with conjunctive radiotherapy and chemotherapy. Malignant gliomas have heterogeneity of tissue-associated antigens (TAAs). To find universal common antigens through different kinds of tumor culture may be the essential issue for tumor vaccine development in the future.

Extracellular pH change modulates the exon 7 splicing in SMN2 mRNA.

Spinal muscular atrophy (SMA) is caused by homozygous deletions/mutations of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene. A nucleotide change in SMN2 results in exon 7 exclusion in the majority of SMN2 mRNA, thus producing low level of SMN protein. Extracellular pH change has been shown to modulate alternative splicing of several pre-mRNAs. In this study, we showed that extracellular pH change can also modulate SMN2 exon 7 splicing in SMA cells. Low extracellular pH enhances SMN2 exon 7 skipping, whereas high extracellular pH promotes its inclusion. Low extracellular pH also reduces SMN protein expression but increases hnRNP A1 expression. In addition, we tested whether intracellular pH-modulating genes could be the modifier of SMA in a SMA discordant family and found that the mRNA levels of ATP6V1B2 gene are significantly higher in two affected siblings than the unaffected one. In conclusion, our results suggest that extracellular pH change modulates SMN2 exon 7 splicing through regulation of hnRNP A1 expression in SMA cells.

5-(N-ethyl-N-isopropyl)-amiloride enhances SMN2 exon 7 inclusion and protein expression in spinal muscular atrophy cells.

OBJECTIVE: Spinal muscular atrophy (SMA) is a common inherited neuromuscular disorder caused by homozygous loss of function of the survival motor neuron 1 (SMN1) gene. All SMA patients carry at least one copy of a nearly identical SMN2 gene. However, a critical nucleotide change in SMN2 results in alternative splicing and exclusion of exon 7 in the majority of SMN2 messenger RNA (mRNA), thus producing a low level of functional SMN protein. Increasing SMN protein production by promoting SMN2 exon 7 inclusion could be a therapeutic approach for SMA. It has been shown that cellular pH microenvironment can modulate pre-mRNA alternative splicing in vivo. In this study, we tested whether inhibitors of the Na+/H+ exchanger can modulate the exon 7 splicing of SMN2 mRNA METHODS: We treated SMA lymphoid cell lines with Na+/H+ exchanger inhibitors and then measured SMN2 exon 7 splicing by reverse transcriptase polymerase chain reaction and SMN protein production by Western blotting and immunofluorescence RESULTS: We found that treatment with an Na+/H+ exchanger inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), significantly enhances SMN2 exon 7 inclusion and SMN protein production in SMA cells. In addition, EIPA increases the number of nuclear gems in SMA cells. We further explored the underlying mechanism, and our results suggest that EIPA may promote SMN2 exon 7 inclusion through upregulation of the splicing factor SRp20 in the nucleus INTERPRETATION: Our finding that EIPA, an inhibitor of the Na+/H+ exchanger, can increase SMN protein expression in SMA cells provides a new direction for the development of drugs for SMA treatment. However, further translational studies are needed to determine whether this finding is applicable for SMA treatment or just a proof of cellular pH effect on SMN splicing.

Flow cytoenzymology of intracellular tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 x 10(5)/100 micro l test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia.