Shawn E. Lupold

miR-21: an androgen receptor-regulated microRNA that promotes hormone-dependent and hormone-independent prostate cancer growth.

Androgen receptor (AR)-mediated oncogenic pathways have not been fully elucidated. In this study, we used high-throughput microarray analysis on two AR-positive prostate cancer (CaP) cell lines to identify 16 AR-responsive microRNAs (miRNA). We focused on miR-21 because of its previously reported oncogenic activity in other cancers. We show androgen-induced AR binding to the defined miR-21 promoter, miPPR-21, suggesting direct transcriptional regulation. Inhibition of miR-21 diminished androgen-induced CaP cell proliferation, providing new evidence that miRNAs can contribute to androgen-driven cell growth. Elevated expression of miR-21 enhanced CaP tumor growth in vivo and, surprisingly, was sufficient for androgen-dependent tumors to overcome castration-mediated growth arrest. Thus, elevated miR-21 expression alone is sufficient to impart castration resistance. Moreover, quantitative reverse transcription-PCR analysis revealed elevated miR-21 expression in CaP when compared with adjacent normal tissue. These results suggest that miR-21 may contribute to CaP pathogenesis.

p53-Induced Up-Regulation of MnSOD and GPx but not Catalase Increases Oxidative Stress and Apoptosis

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca2+ from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

Nucleic acid aptamers: clinical applications and promising new horizons.

Aptamers are a special class of nucleic acid molecules that are beginning to be investigated for clinical use. These small RNA/DNA molecules can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets; they are essentially a chemical equivalent of antibodies. Aptamers have the advantage of being highly specific, relatively small in size, and non-immunogenic. Since the discovery of aptamers in the early 1990s, great efforts have been made to make them clinically relevant for diseases like cancer, HIV, and macular degeneration. In the last two decades, many aptamers have been clinically developed as inhibitors for targets such as vascular endothelial growth factor (VEGF) and thrombin. The first aptamer based therapeutic was FDA approved in 2004 for the treatment of age-related macular degeneration and several other aptamers are currently being evaluated in clinical trials. With advances in targeted-therapy, imaging, and nanotechnology, aptamers are readily considered as potential targeting ligands because of their chemical synthesis and ease of modification for conjugation. Preclinical studies using aptamer-siRNA chimeras and aptamer targeted nanoparticle therapeutics have been very successful in mouse models of cancer and HIV. In summary aptamers are in several stages of development, from pre-clinical studies to clinical trials and even as FDA approved therapeutics. In this review, we will discuss the current state of aptamers in clinical trials as well as some promising aptamers in pre-clinical development.

Synthesis and Evaluation of Technetium-99m- and Rhenium-Labeled Inhibitors of the Prostate-Specific Membrane Antigen (PSMA)

The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging and therapy of cancer. We prepared seven (99m)Tc/Re-labeled compounds by attaching known Tc/Re chelating agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length linker moiety. K i values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the lowest PSMA inhibitory potency of this series, [(99m)Tc(CO)3( L1)] (+) ( L1 = (2-pyridylmethyl)2N(CH2) 4CH(CO2H)NHCO-(CH2) 6CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 +/- 4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the steric requirements of (99m)Tc/Re complexes within PSMA inhibitors, the best results achieved with a linker moiety between the epsilon amine of the urea lysine and the chelator.

Radiohalogenated Prostate-Specific Membrane Antigen (PSMA)- Based Ureas as Imaging Agents for Prostate Cancer

To extend our development of new imaging agents targeting the prostate-specific membrane antigen (PSMA), we have used the versatile intermediate 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid (Lys-C(O)-Glu), which allows ready incorporation of radiohalogens for single photon emission computed tomography (SPECT) and positron emission tomography (PET). We prepared 2-[3-[1-carboxy-5-(4-[(125)I]iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([(125)I]3), 2-[3-[1-carboxy-5-(4-[(18)F]fluoro-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([(18)F]6), and 2-(3-[1-carboxy-5-[(5-[(125)I]iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-pentanedioic acid ([(125)I]8) in 65-80% (nondecay-corrected), 30-35% (decay corrected), and 59-75% (nondecay-corrected) radiochemical yields. Compound [(125)I]3 demonstrated 8.8 +/- 4.7% injected dose per gram (%ID/g) within PSMA(+) PC-3 PIP tumor at 30 min postinjection, which persisted, with clear delineation of the tumor by SPECT. Similar tumor uptake values at early time points were demonstrated for [(18)F]6 (using PET) and [(125)I]8. Because of the many radiohalogenated moieties that can be attached via the epsilon amino group, the intermediate Lys-C(O)-Glu is an attractive template upon which to develop new imaging agents for prostate cancer.

Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts

Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to noncancerous tissues. Radiosensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery, these agents will also sensitize surrounding normal tissues. Here we describe the development of prostate-targeted RNAi agents that selectively sensitized prostate-specific membrane antigen-positive (PSMA-positive) cells to IR. siRNA library screens identified DNA-activated protein kinase, catalytic polypeptide (DNAPK) as an ideal radiosensitization target. DNAPK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNAPK in PCa cells, xenografts, and human prostate tissues. Aptamer-targeted DNAPK shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. These findings support aptamer-shRNA chimeras as selective sensitizing agents for the improved treatment of high-risk localized PCa.

The transcriptional regulation of miR-21, its multiple transcripts, and their implication in prostate cancer

MicroRNAs (miRNAs) are a natural part of the most recently discovered and global regulatory pathway known as RNA interference. Functional studies have shown how specific miRNAs can function as tumor suppressors or oncogenes and, correspondingly, deregulated miRNA profiles have been observed in prostate and other cancers. However, the upstream pathways which regulate miRNA expression are only currently being uncovered. The Androgen Receptor (AR) is a nuclear hormone receptor and transcription factor which plays a paramount role in prostate cancer pathobiology. We performed high throughput miRNA microarray analysis on two AR-responsive cell lines to identified 16 candidate AR-regulated miRNAs. One of the most androgen-induced candidates was a known oncogenic miRNA, miR-21. In a small study of early grade Prostate cancer (PCa) samples we found that miR-21 levels were frequently elevated in comparison to adjacent normal tissue. This observation was supported in the literature and suggests clinical relevance. We found that the activated AR directly interacts with miR-21 regulatory regions, indicating direct transcriptional induction. Furthermore, we provide new reporter studies supporting AR-regulation. Importantly, in functional studies, we found that a modest over-expression of miR-21 enhanced tumor xenograft growth and was sufficient to support androgen-independent proliferation following surgical castration. Thus, our studies suggest a model where miR-21 contributes to androgen-dependent and androgen-independent PCa growth. However, the AR is only one of many reported transcriptional regulators of miR-21. Here we review our recent discoveries and further analyze the reported miR-21 regulatory regions, inhibitory and stimulatory signaling pathways, and primary transcripts.

Sequential SPECT and Optical Imaging of Experimental Models of Prostate Cancer with a Dual Modality Inhibitor of the Prostate-Specific Membrane Antigen

We describe a platform for dual modality (radionuclide/optical) imaging of prostate cancer (PCa) based on targeting the prostate-specific membrane antigen (PSMA). An example provided demonstrates that after a single intravenous (IV) injection of tracer amounts (0.1 nmol) of imaging agent to a tumor-bearing mouse, both single photon emission computed tomography (SPECT) and near-infrared fluorescence (NIRF) imaging were capable of delineating tumor specifically. That such small injected amounts could identify tumor in vivo suggests that optical agents, as has long been known for radiopharmaceuticals, may obey the tracer principle, enabling more rapid clinical translation.

A novel source for miR-21 expression through the alternative polyadenylation of VMP1 gene transcripts

miR-21 is the most commonly over-expressed microRNA (miRNA) in cancer and a proven oncogene. Hsa-miR-21 is located on chromosome 17q23.2, immediately downstream of the vacuole membrane protein-1 (VMP1) gene, also known as TMEM49. VMP1 transcripts initiate ∼130 kb upstream of miR-21, are spliced, and polyadenylated only a few hundred base pairs upstream of the miR-21 hairpin. On the other hand, primary miR-21 transcripts (pri-miR-21) originate within the last introns of VMP1, but bypass VMP1 polyadenylation signals to include the miR-21 hairpin. Here, we report that VMP1 transcripts can also bypass these polyadenylation signals to include miR-21, thus providing a novel and independently regulated source of miR-21, termed VMP1–miR-21. Northern blotting, gene-specific RT-PCR, RNA pull-down and DNA branching assays support that VMP1–miR-21 is expressed at significant levels in a number of cancer cell lines and that it is processed by the Microprocessor complex to produce mature miR-21. VMP1 and pri-miR-21 are induced by common stimuli, such as phorbol-12-myristate-13-acetate (PMA) and androgens, but show differential responses to some stimuli such as epigenetic modifying agents. Collectively, these results indicate that miR-21 is a unique miRNA capable of being regulated by alternative polyadenylation and two independent gene promoters.

Valproic acid inhibits invasiveness in bladder cancer but not in prostate cancer cells.

Histone deacetylase inhibitors (HDACIs) represent a promising new class of antineoplastic agents that affect proliferation, differentiation, and apoptosis in both solid and hematologic malignancies. In addition, HDACIs can alter the expression of at least one cellular adhesion molecule, the coxsackie and adenovirus receptor, in bladder cancer. Because HDACIs can increase expression of a known cellular adhesion molecule, we hypothesized that migration and/or invasion may also be affected. We evaluated this hypothesis using valproic acid (VPA), a commonly prescribed anticonvulsant recently shown to have potent HDACI activity, in the bladder cancer cell lines T24 TCC-SUP, HT1376, and RT4. Analyses of cell migration and invasion were both qualitative (fluorescent microscopy) and quantitative (static and dynamic migration/invasion assays). Our results show that acute VPA treatment (72 h) causes a dose-dependent decrease in invasion for all bladder cancer cell lines, except RT4, a noninvasive papilloma. Migration, in contrast, was not affected by VPA treatment. The inhibitory effect of VPA may be cancer type-specific, because there was no difference in invasion between treated and untreated prostate cancer cell lines LNCaP, PC3, and DU145. Furthermore, when administered chronically (34 days), VPA significantly inhibits growth of T24t tumor xenografts. Our data suggest that VPA exerts some of its antineoplastic effects by inhibiting invasion as well as tumor growth, and thus it may represent a novel adjuvant strategy for patients at high risk of recurrence and/or progression of muscle invasive bladder cancer.