Assembly of therapeutic pRNA-siRNA nanoparticles using bipartite approach.

The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles.

Suppression of Breast Cancer by Small Molecules That Block the Prolactin Receptor.

Prolactin (PRL) is a protein hormone which in humans is secreted by pituitary lactotrophs as well as by many normal and malignant non-pituitary sites. Many lines of evidence demonstrate that both circulating and locally produced PRL increase breast cancer (BC) growth and metastases and confer chemoresistance. Our objective was to identify and then characterize small molecules that block the tumorigenic actions of PRL in BC. We employed three cell-based assays in high throughput screening (HTS) of 51,000 small molecules and identified two small molecule inhibitors (SMIs), named SMI-1 and SMI-6. Both compounds bound to the extracellular domain (ECD) of the PRL receptor (PRLR) at 1-3 micromolar affinity and abrogated PRL-induced breast cancer cell (BCC) invasion and malignant lymphocyte proliferation. SMI-6 effectively reduced the viability of multiple BCC types, had much lower activity against various non-malignant cells, displayed high selectivity, and showed no apparent in vitro or in vivo toxicity. In athymic nude mice, SMI-6 rapidly and dramatically suppressed the growth of PRL-expressing BC xenografts. This report represents a pre-clinical phase of developing novel anti-cancer agents with the potential to become effective therapeutics in breast cancer patients.

Bisphenol A at low nanomolar doses confers chemoresistance in estrogen receptor-alpha-positive and -negative breast cancer cells.

BACKGROUND: Resistance to chemotherapy is a major problem facing breast cancer patients, and identifying potential contributors to chemoresistance is a critical area of research. Bisphenol A (BPA) has long been suspected to promote carcinogenesis, but the high doses of BPA used in many studies generated conflicting results. In addition, the mechanism by which BPA exerts its biological actions is unclear. Although estrogen has been shown to antagonize anticancer drugs, the role of BPA in chemoresistance has not been examined. OBJECTIVE: The objective of our study was to determine whether BPA at low nanomolar concentrations opposes the action of doxorubicin, cisplatin, and vinblastine in the estrogen receptor-alpha (ERalpha)-positive T47D and the ERalpha-negative MDA-MB-468 breast cancer cells. METHODS: We determined the responsiveness of cells to anticancer drugs and BPA using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Specific ERalpha and ERbeta inhibitors and real-time polymerase chain reaction were used to identify potential receptor(s) that mediate the actions of BPA. Expression of antiapoptotic proteins was assessed by Western blotting. RESULTS: BPA antagonizes the cytotoxicity of multiple chemotherapeutic agents in both ERalpha-positive and -negative breast cancer cells independent of the classical ERs. Both cell types express alternative ERs, including G-protein-coupled receptor 30 (GPR30) and members of the estrogen-related receptor family. Increased expression of antiapoptotic proteins is a potential mechanism by which BPA exerts its anticytotoxic effects. CONCLUSIONS: BPA at environmentally relevant doses reduces the efficacy of chemotherapeutic agents. These data provide considerable support to the accumulating evidence that BPA is hazardous to human health.

Liver-specific pRB loss results in ectopic cell cycle entry and aberrant ploidy.

The liver exhibits an exquisitely controlled cell cycle, wherein hepatocytes are maintained in quiescence until stimulated to proliferate. The retinoblastoma tumor suppressor, pRB, plays a central role in proliferative control by inhibiting inappropriate cell cycle entry. In many cases, liver cancer arises due to aberrant cycles of proliferation, and correspondingly, pRB is functionally inactivated in the majority of hepatocellular carcinomas. Therefore, to determine how pRB loss may provide conditions permissive for deregulated hepatocyte proliferation, we investigated the consequence of somatic pRB inactivation in murine liver. We show that liver-specific pRB loss results in E2F target gene deregulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets are repressed and hepatocytes become quiescent independent of pRB, suggesting that other factors may compensate for pRB loss. Therefore, to probe the consequences of acute pRB inactivation in livers of adult mice, we gave adenoviral-Cre by i.v. injection. We show that acute pRB loss is sufficient to elicit E2F target gene expression and cell cycle entry in adult liver, demonstrating a critical role for pRB in maintaining hepatocyte quiescence. Finally, we show that liver-specific pRB loss results in the development of nuclear pleomorphism associated with elevated ploidy that is evident in adult mice harboring both acute and chronic pRB loss. Together, these results show the crucial role played by pRB in maintaining hepatocyte quiescence and ploidy in adult liver in vivo and underscore the critical importance of delineating the consequences of acute pRB loss in adult animals.

The cyclic GMP/protein kinase G pathway as a therapeutic target in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with high mortality. Treatments, which can result in significant morbidity, have not substantially changed in three decades. The second messenger cyclic GMP (cGMP), which targets protein kinase G (PKG), is generated by guanylate cyclases (GCs), and is rapidly hydrolyzed by phosphodiesterases (PDEs). Activation of the cGMP/PKG pathway is antineoplastic in several cancer types, but its impact on HNSCC has not been fully exploited. We found differential expression of critical components of this pathway in four HNSCC cell lines. Several activators of soluble GC (sGC), as well as inhibitors of PDE5, increased intracellular cGMP, reduced cell viability, and induced apoptosis in HNSCC cells. The apoptotic effects of the sGC activator BAY 41-2272 and the PDE5 inhibitor Tadalafil (Cialis) were mediated by PKG. Furthermore, Tadalafil substantially reduced the growth of CAL27-derived tumors in athymic mice. Several drugs which either activate sGC or inhibit PDE5 are approved for treatment of nonmalignant conditions. These drugs could be repurposed as novel and effective therapeutics in patients with head and neck cancer.

SWI/SNF activity is required for the repression of deoxyribonucleotide triphosphate metabolic enzymes via the recruitment of mSin3B.

The SWI/SNF chromatin remodeling complex plays a critical role in the coordination of gene expression with physiological stimuli. The synthetic enzymes ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase are coordinately regulated to ensure appropriate deoxyribonucleotide triphosphate levels. Particularly, these enzymes are actively repressed as cells exit the cell cycle through the action of E2F transcription factors and the retinoblastoma tumor suppressor/p107/p130 family of pocket proteins. This process is found to be highly dependent on SWI/SNF activity as cells deficient in BRG-1 and Brm subunits fail to repress these genes with activation of pocket proteins, and this deficit in repression can be complemented, via the ectopic expression of BRG-1. The failure to repress transcription does not involve a blockade in the association of E2F or pocket proteins p107 and p130 with promoter elements. Rather, the deficit in repression is due to a failure to mediate histone deacetylation of ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase promoters in the absence of SWI/SNF activity. The basis for this is found to be a failure to recruit mSin3B and histone deacetylase proteins to promoters. Thus, the coordinate repression of deoxyribonucleotide triphosphate metabolic enzymes is dependent on the action of SWI/SNF in facilitating the assembly of repressor complexes at the promoter.

Loss of RB compromises specific heterochromatin modifications and modulates HP1alpha dynamics.

Heterochromatin domains are important for gene silencing, centromere organization, and genomic stability. These genomic domains are marked with specific histone modifications, heterochromatin protein 1 (HP1) binding and DNA methylation. The retinoblastoma tumor suppressor, RB mediates transcriptional repression and functionally interacts with a number of factors that are involved in heterochromatin biology including HP1, Suv39h1, DNMT1, and components of the SWI/SNF chromatin remodeling complex. To analyze the specific influence of RB loss on chromatin modification, mouse adult fibroblasts (MAFs) derived from Rb(loxP/loxP) mice were utilized to acutely knockout RB. In this setting, target genes of RB are deregulated. Additionally, changes in histone modifications were observed. Specifically, histone H4 lysine 20 trimethylation was absent from heterochromatin domains following loss of RB and there were changes in the relative levels of histone modifications between RB-proficient and deficient cells. While RB loss significantly altered the modifications associated with heterochromatin domains, these domains were readily identified and efficiently mediated the recruitment of HP1alpha. Kinetic analyses of HP1alpha within the heterochromatin domains present in RB-deficient cells indicated that loss of RB retarded HP1alpha dynamics, indicating that HP1alpha is paradoxically more tightly associated with heterochromatin in the absence of RB function. Combined, these analyses demonstrate that loss of RB has global effects on chromatin modifications and dynamics.

RB loss abrogates cell cycle control and genome integrity to promote liver tumorigenesis.

BACKGROUND & AIMS: The retinoblastoma (RB) tumor suppressor is functionally inactivated in most hepatocellular carcinomas (HCC), although the mechanisms by which RB suppresses liver tumorigenesis are poorly defined. We investigated the impact of RB loss on carcinogen-induced liver tumorigenesis. METHODS: Mice harboring liver-specific RB ablation and normal littermates were exposed to the hepatocarcinogen diethylnitrosamine (DEN). The influence of RB loss on liver tumorigenesis was assessed by evaluating tumor multiplicity, proliferation, and genome integrity within tumors arising in RB-deficient and wild-type livers. In silico analyses were used to probe the association between gene expression signatures for RB loss and chromosomal instability and the ability of genes up-regulated by RB loss to predict the survival of human HCC patients. RESULTS: RB deficiency significantly increased tumor multiplicity in livers exposed to DEN. Although hepatocytes in nontumor regions of DEN-exposed livers were quiescent regardless of RB status, tumors arising in RB-deficient livers were significantly more proliferative than those in normal livers and expressed high levels of RB/E2F target genes. Analysis of genes up-regulated by RB loss demonstrated significant overlap with a gene expression signature associated with chromosomal instability. Correspondingly, tumors arising in RB-deficient livers were significantly more likely to harbor hepatocytes exhibiting altered ploidy. Finally, gene expression analysis of human HCCs demonstrated that elevated expression of RB-regulated genes independently predicts poor survival. CONCLUSIONS: RB deletion in the mouse liver enhances DEN-induced tumorigenesis, associated with increased hepatocyte proliferation and compromised genome integrity. Evaluation of RB status may be a useful prognostic factor in human HCC.

Cyclin D1 splice variants. Differential effects on localization, RB phosphorylation, and cellular transformation.

Cyclin D1 is a proto-oncogene that functions by inactivation of the retinoblastoma tumor suppressor protein, RB. A common polymorphism in the cyclin D1 gene is associated with the production of an alternate transcript of cyclin D1, termed cyclin D1b. Both the polymorphism and the variant transcript are associated with increased risk for multiple cancers and the severity of a given cancer; however, the underlying activities of cyclin D1b have not been elucidated relative to the canonical cyclin D1a. Because cyclin D1b does not possess the threonine 286 phosphorylation site required for nuclear export and regulated degradation, it has been hypothesized to encode a stable nuclear protein that would constitutively inactivate the RB pathway. Surprisingly, we find that cyclin D1b protein does not inappropriately accumulate in cells and exhibits stability comparable to cyclin D1a. As expected, the cyclin D1b protein was constitutively localized in the nucleus, whereas cyclin D1a was exported to the cytoplasm in S-phase. Despite enhanced nuclear localization, we find that cyclin D1b is a poor catalyst of RB phosphorylation/inactivation. However, cyclin D1b potently induced cellular transformation in contrast to cyclin D1a. In summary, we demonstrate that cyclin D1b specifically disrupts contact inhibition in a manner distinct from cyclin D1a. These data reveal novel roles for d-type cyclins in tumorigenesis.