Identification of thiostrepton as a novel therapeutic agent that targets human colon cancer stem cells.

Accumulating evidence shows that colorectal cancer stem cells (CRSCs) are largely responsible for the metastasis and relapse of colorectal cancer (CRC) after therapy. Hence, identifying new agents that specifically target CRSCs would help improve the effectiveness of current CRC therapies. To accelerate identification of agents targeting CRSCs, the Connectivity Map (CMap) approach was used. Among the top-ranked candidates, thiostrepton, a thiazole antibiotic, was selected for further investigation because of its known tumoricidal activity. Thiostrepton could selectively induce apoptosis in CRSC subpopulations in both parental HCT-15 and HT-29 human CRC lines as well as in EMT and chemoresistant clones derived from them. Further, we investigated its inhibitory effects on the sphere- and colony-forming capabilities of the aforementioned CRC lines. The in vitro inhibition of sphere and colony formation was associated with downregulation of various modulators of the stem cell phenotype. The combination of thiostrepton and oxaliplatin eradicated both CD44(+) HCT-15 and HT-29 cells more efficiently than either drug alone. FoxM1, an oncogenic transcription factor, was identified as a critical positive modulator of stemness and as the main target of thiostrepton in the CRC lines. This is the first report showing the selective killing of CRSCs by thiostrepton, which has been proposed to be a promising anti-neoplastic agent. On the basis of its synergism with oxaliplatin in killing CRSCs in vitro, if this activity is confirmed in vivo, thiostrepton may be a promising agent to be used clinically in combination with current chemotherapies to improve the efficacy of these regimens.

Identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data.

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis, tumor maintenance and therapeutic resistance. Thus, to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse, at least in part, the gene expression signature of GBM and GSCs, this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here, we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened, thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine, we found that thioridazine induces autophagy in GBM cell lines, and upregulates AMPK activity. Moreover, LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition, thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent, but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties.

Identification of latent biomarkers in hepatocellular carcinoma by ultra-deep whole-transcriptome sequencing.

There is an urgent need to identify biomarkers for hepatocellular carcinoma due to limited treatment options and the poor prognosis of this common lethal disease. Whole-transcriptome shotgun sequencing (RNA-Seq) provides new possibilities for biomarker identification. We sequenced ∼250 million pair-end reads from a pair of adjacent normal and tumor liver samples. With the aid of bioinformatics tools, we determined the transcriptome landscape and sought novel biomarkers by further empirical validations in 55 pairs of adjacent normal and tumor liver samples with various viral statuses such as HBV(+), HCV(+) and HBV(-)HCV(-). We identified a novel gene with coding regions, termed DUNQU1, which has a tissue-specific expression pattern in tumor liver samples of HCV(+) and HBV(-)HCV(-) hepatocellular carcinomas. Overexpression of DUNQU1 in Huh7 cell lines enhances the ability to form colonies in soft agar. Also, we identified three novel differentially-expressed protein-coding genes (ALG1L, SERPINA11 and TMEM82) that lack documented expression profiles in liver cancer and showed that the level of SREPINA11 is correlated with pathology stages. Moreover, we showed that the alternative splicing event of FGFR2 is associated with virus infection, tumor size, cirrhosis and tumor recurrence. The findings indicate that these new markers of hepatocellular carcinoma may be of value in improving prognosis and could have potential as new targets for developing new treatment options.

Malignant pleural effusion cells show aberrant glucose metabolism gene expression.

Malignant pleural effusion (MPE) accompanying lung adenocarcinoma indicates poor prognosis and early metastasis. This study aimed to identify genes related to MPE formation. Three tissue sample cohorts, seven from healthy lungs, 18 from stage I-III lung adenocarcinoma with adjacent healthy lung tissue and 13 from lung adenocarcinomas with MPE, were analysed by oligonucleotide microarray. The identified genes were verified by quantitative real-time PCR (qRT-PCR), immunohistochemical staining, and immunofluorescence confocal microscopy. 20 up- or down-regulated genes with a two-fold change in MPE cancer cells compared to healthy tissues were differentially expressed from early- to late-stage lung cancer. Of 13 genes related to cellular metabolism, aldolase A (ALDOA), sorbitol dehydrogenase (SORD), transketolase (TKT), and tuberous sclerosis 1 (TSC1) were related to glucose metabolism. qRT-PCR validated their mRNA expressions in pleural metastatic samples. Immunohistochemical staining confirmed aberrant TKT, ALDOA, and TSC1 expressions in tumour cells. Immunofluorescence confirmed TKT co-localisation and co-distribution of ALDOA with thyroid transcription factor 1-positive cancer cells. TKT regulated the proliferation, vascular endothelial growth factor secretion in vitro and in vivo vascular permeability of cancer cell. Glucose metabolic reprogramming by ALDOA, SORD, TKT and TSC1 is important in MPE pathogenesis.

Expression of FLJ10540 is correlated with aggressiveness of oral cavity squamous cell carcinoma by stimulating cell migration and invasion through increased FOXM1 and MMP-2 activity.

Matrix metalloproteinase (MMP)-2 plays critical roles in tumor development and in the metastasis of multiple cancers, including human oral cavity squamous cell carcinoma (OCSCC). One of the upstream regulators of MMP-2 is FOXM1, which is overexpressed in a microarray dataset of OCSCC. It is interesting that FLJ10540 exhibits similar gene expression profiles with MMP-2 and FOXM1, raising the possibility that these molecules might participate in MMP-2-elicited cancer progression and metastasis of OCSCC. To examine this connection, we first showed that FLJ10540 was significantly overexpressed in OCSCC. A strong FLJ10540 expression was significantly correlated with an advanced tumor node metastasis stage and the cumulative 5-year survival rate. Thus, an elevated FLJ10540 expression is an indicator of poor survival. Functionally, FLJ10540 had the abilities to stimulate cell migration and invasion in oral cancer cells through increased FOXM1 and MMP-2 expressions. Conversely, the depletion of the FLJ10540 expression by small interfering RNAs suppressed the FOXM1 and MMP-2 protein expressions. The suppression of either FLJ10540 or FOXM1 could cause significant inhibition on cell migratory and invasive ability in oral cancer cells. Finally, the immunohistochemical and western blotting analyses of human aggressive OCSCC specimens showed a significant positive correlation among FLJ10540, FOXM1 and MMP-2 expressions. These findings suggest that FLJ10540 is not only an important prognostic factor but also a new therapeutic target in the FLJ10540/FOXM1/MMP-2 pathway for OCSCC treatment.

FLJ10540-elicited cell transformation is through the activation of PI3-kinase/AKT pathway.

A significant challenge in the post-genomic era is how to prioritize differentially expressed and uncharacterized novel genes found in hepatocellular carcinoma (HCC) microarray profiling. One such category is cell cycle regulated genes that have only evolved in higher organisms but not in lower eukaryotic cells. Characterization of these genes may reveal some novel human cancer-specific abnormalities. A novel transcript, FLJ10540 was identified. FLJ10540 is overexpressed in HCC as examined by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. The patients with higher FLJ10540 expression had a poor survival than those with lower FLJ10540 expression. Functional characterization indicates that FLJ10540 displays a number of characteristics associated with an oncogene, including anchorage-independent growth, enhanced cell growth at low serum levels and induction of tumorigenesis in nude mice. FLJ10540-elicited cell transformation is mediated by activation of the phosphatidylinositol 3'-kinase (PI3K)/AKT pathway. Moreover, FLJ10540 forms a complex with PI3K and can activate PI3K activity, which provides a mechanistic basis for FLJ10540-mediated oncogenesis. Together, using a combination of bioinformatics searches and empirical data, we have identified a novel oncogene, FLJ10540, which is conserved only in higher organisms. The finding raises the possibility that FLJ10540 is a potential new therapeutic target for HCC treatment. These findings may contribute to the development of new therapeutic strategies that are able to block the PI3K/AKT pathway in cancer cells.

Caspase 3, periodically expressed and activated at G2/M transition, is required for nocodazole-induced mitotic checkpoint.

Caspases have been known for several years for their involvement in executing apoptosis, where unwanted or damaged cells are eliminated. Surprisingly, after analysis of the relevant data set from the Stanford microarray database, we noticed that the gene expression pattern for caspase 3, but not for caspase 1, 6, 7, 8, 9, or 10, undergoes periodic change in the HeLa cell cycle. In this study, we have demonstrated that caspase 3, but not other caspases, is upregulated and activated just prior to mitosis. Pretreatment of human hepatoma cells with a caspase 3 inhibitor z-DEVD-FMK, prior to the treatment with an antimicrotubule drug nocodazole, abrogates the mitotic arrest, suggesting that caspase 3 (or a caspase 3-like enzyme) might be involved in mitotic-spindle checkpoint. The studies not only characterize caspase 3 as a cell cycle-regulated protein, but also link the protein to nocodazole-dependent mitotic checkpoint, greatly expanding the understanding of caspase 3.