Dehua Yu

Nuclear Hormone Receptor NR4A2 Is Involved in Cell Transformation and Apoptosis

HeLaHF cells are transformation revertants of cervical cancer HeLa cells and have lost anchorage-independent growth potential and tumorigenicity. Activation of tumor suppressor(s) was implicated previously in this transformation reversion. In this study, expression profiling analysis was carried out to identify potential oncogenes that are down-regulated in HeLaHF cells. We found that all three members of the NR4A1/Nur77/NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) were down-regulated in the HeLaHF revertant. Small interfering RNA-mediated down-regulation of NR4A2 in HeLa cells, either transiently or stably, resulted in reduced anchorage-independent growth that was largely attributable to increased anoikis. Furthermore, down-regulation of NR4A2 as well as NR4A1 promoted intrinsic apoptosis. These phenotypes were also observed in several other experimental cancer cells, suggesting the observed apoptosis suppression is a more general property of NR4A2 and NR4A1. These phenotypes also suggest that the Nur77/NGFIB subfamily of orphan receptors exhibit certain oncogenic functionalities with regards to cell proliferation and apoptosis and could therefore be evaluated as potential cancer therapeutic targets.

One-week, 96-well soft agar growth assay for cancer target validation

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of todays oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

Pyrvinium Targets the Unfolded Protein Response to Hypoglycemia and Its Anti-Tumor Activity Is Enhanced by Combination Therapy

We identified pyrvinium pamoate, an old anthelminthic medicine, which preferentially inhibits anchorage-independent growth of cancer cells over anchorage-dependent growth (∼10 fold). It was also reported by others to have anti-tumor activity in vivo and selective toxicity against cancer cells under glucose starvation in vitro, but with unknown mechanism. Here, we provide evidence that pyrvinium suppresses the transcriptional activation of GRP78 and GRP94 induced by glucose deprivation or 2-deoxyglucose (2DG, a glycolysis inhibitor), but not by tunicamycin or A23187. Other UPR pathways induced by glucose starvation, e.g. XBP-1, ATF4, were also found suppressed by pyrvinium. Constitutive expression of GRP78 via transgene partially protected cells from pyrvinium induced cell death under glucose starvation, suggesting that suppression of the UPR is involved in pyrvinium mediated cytotoxicity under glucose starvation. Xenograft experiments showed rather marginal overall anti-tumor activity for pyrvinium as a monotherapy. However, the combination of pyrvinium and Doxorubicin demonstrated significantly enhanced efficacy in vivo, supporting a mechanistic treatment concept based on tumor hypoglycemia and UPR.

Orphan G protein–coupled receptor GPR56 plays a role in cell transformation and tumorigenesis involving the cell adhesion pathway

GPR56 is an orphan G protein–coupled receptor, mutations of which have recently been associated with bilateral frontoparietal polymicrogyria, a rare neurologic disease that has implications in brain development. However, no phenotype beyond central nervous system has yet been described for the GPR56-null mutations despite abundant GPR56 expression in many non–central nervous system adult tissues. In the present study, we show that higher GPR56 expression is correlated with the cellular transformation phenotypes of several cancer tissues compared with their normal counterparts, implying a potential oncogenic function. RNA interference–mediated GPR56 silencing results in apoptosis induction and reduced anchorage-independent growth of cancer cells via increased anoikis, whereas cDNA overexpression resulted in increased foci formation in mouse fibroblast NIH3T3 cell line. When GPR56 silencing was induced in vivo in several xenograft tumor models, significant tumor responses (including regression) were observed, suggesting the potential of targeting GPR56 in the development of tumor therapies. The expression profiling of GPR56-silenced A2058 melanoma cell line revealed several genes whose expression was affected by GPR56 silencing, particularly those in the integrin-mediated signaling and cell adhesion pathways. The potential role of GPR56 in cancer cell adhesion was further confirmed by the observation that GPR56 silencing also reduced cell adhesion to the extracellular matrix, which is consistent with the observed increase in anoikis and reduction in anchorage-independent growth phenotypes. The oncogenic potential and apparent absence of physiologic defects in adult human tissues lacking GPR56, as well as the targetable nature of G protein–coupled receptor by small molecule or antibody, make GPR56 an attractive drug target for the development of cancer therapies. [Mol Cancer Ther 2007;6(6):1840–50]

A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery

The lentiviral vector is a useful tool for delivery of hairpin siRNA (shRNA) into mammalian cells. However, the efficiency of this system for carrying double-stranded siRNA (dsRNA) has not been explored. In this study we cloned the two forms of siRNA-coding sequence, a palindromic DNA with a spacer loop for shRNA and a double-stranded DNA with opposing Pol III promoters for dsRNA, into lentiviral DNA vectors, and compared their viral vector production yields. Our results indicate that sharply lower titer vector was obtained for dsRNA while much higher titer vector was produced for shRNA, posing a fundamental concern whether siRNA-carrying viral RNA itself is an inherent target of RNAi. Further experimental analyses using packaging cells that either allow or do not allow siRNA transcription indicate that the shRNA-carrying viral RNA is resistant to RNAi but the viral RNA carrier for dsRNA is not, offering a linker of RNAi bias-target secondary structure that causes shRNA vector to evade RNAi degradation. More importantly, the poor yield of dsRNA vector production was restored when a novel packaging cell line was used that blocks the antisense strand from dsRNA duplexes. This method has important implications for the RNAi field, especially for those who are using lentiviral dsRNA and dsRNA libraries for various biological discovery and therapeutic interventions.

Generation of shRNA Pool Library: A Revision of the Biological Technique from the Viewpoint of Chemistry

RNA interference (RNAi) is the process of using a small doublestranded RNA (siRNA) to knock down the expression level of sequence–homologue genes. The selective and robust effect of siRNA on gene expression makes RNAi one of the most important technological breakthroughs in modern scientific history. Short hairpin siRNA (shRNA) is the most widely used form of expressed siRNA in which RNA duplexes are tethered through a small loop. Currently the use of individual shRNA to study gene function is commonplace. The newest trend is to use siRNA/shRNA libraries to perform reverse-genetic screens of hundreds or even thousands of different genes in a single experiment. Three major factors limit the broad application of syntheticarray-based siRNA libraries: the enormous cost of genomewide siRNA libraries and the expensive robotic liquid handling systems for high-throughput screening as well as the difficulties in performing transfections reproducibly. A much easier alternative is to prepare an siRNA library that is first expressed in transduced cells, then the cells that have the desired phenotype are selected, and the siRNA sequence that is contained within the selected cells is identified. Several groups have reported a technique for the construction of such a shRNA pool library. The key step in this technique is to convert a pool of single-stranded hairpin DNA into a pool of doublestranded DNA (shRNA templates) via special DNA polymerase-catalyzed primer extension (Figure 1A) prior to cloning into expression vectors (see Figure S1 in the Supporting Information). Given the enormous advantage of this method, however, no follow-up application has been ACHTUNGTRENNUNGreported. Here, we revise the feasibility of this step from the viewpoint of chemistry. The goal of primer extension is to disrupt intramolecular base pairs within the highly stable hairpin DNA template, and to synthesize a complementary single-stranded DNA as shown in Figure 1A. It should be noted that the newly synthesized single-stranded DNA is not only complementary, but more importantly, sequence-identical to its template, and thus their annealing product is a palindromic DNA, except for the small loop portion. However, the fundamental concern is whether this reaction, that is, the displacement of intramolecular base pairs with intermolecular base pairs, is energetically favorable, or which form in this conversion, hairpin or the double strand, is more stable. In order to clarify this point, we tested the thermostability of palindromic DNA. A pool of double-stranded DNA (the siRNA-