Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from it's action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repression in vitro . In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activity in vivo . Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.
Cancer is one of the most common causes of death worldwide. Two types of cancer that have high mortality rates are pancreatic and lung cancer. Despite improvements in treatment strategies, resistance to chemotherapy and the presence of metastases are common. Therefore, novel therapies which target and silence genes involved in regulating these processes are required. Short-interfering RNA (siRNA) holds great promise as a therapeutic to silence disease-causing genes. However, siRNA requires a delivery vehicle to enter the cell to allow it to silence its target gene. Herein, we report on the design and synthesis of cationic star polymers as novel delivery vehicles for siRNA to silence genes in pancreatic and lung cancer cells. Dimethylaminoethyl methacrylate (DMAEMA) was polymerized via reversible addition-fragmentation transfer polymerization (RAFT) and then chain extended in the presence of both cross-linkers N,N-bis(acryloyl)cistamine and DMAEMA, yielding biodegradable well-defined star polymers. The star polymers were characterized by transmission electron microscopy, dynamic light scattering, ζ potential, and gel permeation chromatography. Importantly, the star polymers were able to self-assemble with siRNA and form small uniform nanoparticle complexes. Moreover, the ratios of star polymer required to complex siRNA were nontoxic in both pancreatic and lung cancer cells. Treatment with star polymer-siRNA complexes resulted in uptake of siRNA into both cell lines and a significant decrease in target gene mRNA and protein levels. In addition, delivery of clinically relevant amounts of siRNA complexed to the star polymer were able to silence target gene expression by 50% in an in vivo tumor setting. Collectively, these results provide the first evidence of well-defined small cationic star polymers to deliver active siRNA to both pancreatic and lung cancer cells and may be a valuable tool to inhibit key genes involved in promoting chemotherapy drug resistance and metastases.
bIII-tubulin (encoded by TUBB3) expression is associated with therapeutic resistance and aggressive disease in non-small cell lung cancer (NSCLC), but the basis for its pathogenic influence is not understood. Functional and differential proteomics revealed that bIII-tubulin regulates expression of proteins associated with malignant growth and metastases. In particular, the adhesionassociated tumor suppressor maspin was differentially regulated by bIII-tubulin. Functionally, bIII-tubulin suppression altered cell morphology, reduced tumor spheroid outgrowth, and increased sensitivity to anoikis. Mechanistically, the PTEN/AKT signaling axis was defined as a critical pathway regulated by bIII-tubulin in NSCLC cells. bIII-Tubulin blockage in vivo reduced tumor incidence and growth. Overall, our findings revealed how bIII-tubulin influences tumor growth in NSCLC, defining new biologic functions and mechanism of action of bIII-tubulin in tumorigenesis.Cancer Res; 75(2); 415-25. Ó2014 AACR.
Non-small cell lung cancer (NSCLC) remains the most common cause of cancer death worldwide due its resistance to chemotherapy and aggressive tumor growth. Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase which is overexpressed in cancer cells, and plays a major role in regulating tumor growth. A number of PLK1 inhibitors are in clinical trial; however, poor tumor bioavailability and off-target effects limit their efficacy. Short-interfering-RNA (siRNA) holds promise as a class of therapeutics, which can selectively silence disease-causing genes. However, siRNA cannot enter cells without a delivery vehicle. Herein, we investigated whether RNAi-interfering nanoparticles could deliver siRNA to NSCLC cells and silence PLK1 expression in vitro and in vivo. iNOP-7 was non-toxic, and delivered siRNA with high efficiency to NSCLC cells. iNOP-7-PLK1 siRNA silenced PLK1 expression and reduced NSCLC growth in vitro. Notably, iNOP-7 delivered siRNA to orthotopic lung tumors in mice, and administration of iNOP-7-PLK1 siRNA reduced lung tumor burden. These novel data show that iNOP-7 can deliver siRNA against PLK1 to NSCLC cells, and decrease cell proliferation both in vitro and in vivo. iNOP-7-PLK1 siRNA may provide a novel therapeutic strategy for the treatment of NSCLC as well as other cancers which aberrantly express this gene.
Poly(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3′-sense or 5′-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3′-sense or 5′-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3′-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells.
BackgroundHistone deacetylase inhibitors (HDACIs) have many effects on cancer cells, such as growth inhibition, induction of cell death, differentiation, and anti-angiogenesis, all with a wide therapeutic index. However, clinical trials demonstrate that HDACIs are more likely to be effective when used in combination with other anticancer agents. Moreover, the molecular basis for the anti-cancer action of HDACIs is still unknown. In this study, we compared different combinations of HDACIs and anti-cancer agents with anti-angiogenic effects, and analysed their mechanism of action.ResultsTrichostatin A (TSA) and α-interferon (IFNα) were the most effective combination across a range of different cancer cell lines, while normal non-malignant cells did not respond in the same manner to the combination therapy. There was a close correlation between absence of basal p21WAF1 expression and response to TSA and IFNα treatment. Moreover, inhibition of p21WAF1 expression in a p21WAF1-expressing breast cancer cell line by a specific siRNA increased the cytotoxic effects of TSA and IFNα. In vitro assays of endothelial cell function showed that TSA and IFNα decreased endothelial cell migration, invasion, and capillary tubule formation, without affecting endothelial cell viability. TSA and IFNα co-operatively inhibited gene expression of some pro-angiogenic factors: vascular endothelial growth factor, hypoxia-inducible factor 1α and matrix metalloproteinase 9, in neuroblastoma cells under hypoxic conditions. Combination TSA and IFNα therapy markedly reduced tumour angiogenesis in neuroblastoma-bearing transgenic mice.ConclusionOur results indicate that combination TSA and IFNα therapy has potent co-operative cytotoxic and anti-angiogenic activity. High basal p21WAF1 expression appears to be acting as a resistance factor to the combination therapy.
The cyclin-dependent kinase inhibitor, p21(WAF1), induces cell-cycle arrest and can act as a tumor suppressor. However, increasing evidence indicates that p21(WAF1) can also increase resistance to some anticancer therapies and thus promote tumor growth. The mechanisms explaining this paradox have not been explained. We found that conditioned media from MCF-7 breast cancer cells transfected with a p21(WAF1)-specific small interfering RNA (siRNA) significantly reduced endothelial cell migration, invasion and vascular sprouting. Liquid chromatography/mass spectrometry analysis of the conditioned media revealed that p21(WAF1) knockdown significantly reduced secretion of thioredoxin (Trx), a redox protein known to promote tumor angiogenesis. p21(WAF1) knockdown decreased Trx enzymatic activity in cancer cells, by effects on the expression levels of intracellular thioredoxin-binding protein 2 (TBP2), known to bind and inactivate Trx. Consistent with these findings, media from cancer cells transfected with TBP2 siRNA promoted endothelial cell invasion and blocked the anti-angiogenic effect of p21(WAF1) siRNA. Addition of Trx siRNA blocked the pro-angiogenic effects of TBP2 siRNA. Chromatin immunoprecipitation assays showed p21(WAF1) bound TBP2 gene promoter. Taken together, our data suggests that p21(WAF1) can induce Trx secretion and angiogenesis in cancer cells, by direct transcriptional repression of the TBP2 promoter.
Facilitating informed decision‐making regarding genetic testing is a core component of genetic counseling practice. Internationally, genetic testing is shifting toward gene panels and genomic testing, including whole exome and whole genome sequencing to improve diagnostic yield and cost‐effectiveness. This study explored genetics practitioners’ current experience with panels and genomic tests and the associated evolution of genetic counseling practice. Genetics practitioners with genomic testing experience, were purposively invited to participate in a semi‐structured telephone interview and to snowball the invitation to colleagues. Interviews conducted with participants residing in Australia (n = 9) and the UK (n = 5) were transcribed and analyzed using an inductive thematic approach. Three themes emerged: (a) Role delineation: current roles, future roles, and the influence of increasing complexity; (b) The evolving spectrum of practice: blurred boundaries between research and clinical services; impact on facilitation of informed consent; and return of results strategies; and (c) Policy and governance needs: equality of access; achieving consistent variant interpretation, reporting, and responsibility for review; managing incidental findings; and professional regulation for Australian genetic counselors. These exploratory data highlight that genetic counseling practice and the essential role of facilitating informed consent are evolving but remain patient‐centered, with core skills underpinning practitioners’ capacity to adapt.
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