DNA vaccination for cervical cancer; a novel technology platform of RALA mediated gene delivery via polymeric microneedles.

HPV subtypes (16, 18) are associated with the development of cervical cancer, with oncoproteins E6 and E7 responsible for pathogenesis. The goal of this study was to evaluate our 'smart system' technology platform for DNA vaccination against cervical cancer. The vaccination platform brings together two main components; a peptide RALA which condenses DNA into cationic nanoparticles (NPs), and a polymeric polyvinylpyrrolidone (PVP) microneedle (MN) patch for cutaneous delivery of the loaded NPs. RALA condensed E6/E7 DNA into NPs not exceeding 100nm in diameter, and afforded the DNA protection from degradation in PVP. Sera from mice vaccinated with MN/RALA-E6/E7 were richer in E6/E7-specific IgGs, displayed a greater T-cell-mediated TC-1 cytotoxicity and contained more IFN-γ than sera from mice that received NPs intramuscularly. More importantly, MN/RALA-E6/E7 delayed TC-1 tumor initiation in a prophylactic model, and slowed tumor growth in a therapeutic model of vaccination, and was more potent than intramuscular vaccination.

Potentiating the Anticancer Properties of Bisphosphonates by Nanocomplexation with the Cationic Amphipathic Peptide, RALA.

Bisphosphonates (BPs) are a class of bone resorptive drug with a high affinity for the hydroxyapatite structure of bone matrices that are used for the treatment of osteoporosis. However, clinical application is limited by a common toxicity, BP-related osteonecrosis of the jaw. There is emerging evidence that BPs possess anticancer potential, but exploitation of these antiproliferative properties is limited by their toxicities. We previously reported the utility of a cationic amphipathic fusogenic peptide, RALA, to traffic anionic nucleic acids into various cell types in the form of cationic nanoparticles. We hypothesized that complexation with RALA could similarly be used to conceal a BP's hydroxyapatite affinity, and to enhance bioavailability, thereby improving anticancer efficacy. Incubation of RALA with alendronate, etidronate, risedronate, or zoledronate provoked spontaneous electrostatic formation of cationic nanoparticles that did not exceed 100 nm in diameter and that were stable over a range of temperatures and for up to 6 h. The nanoparticles demonstrated a pH responsiveness, possibly indicative of a conformational change, that could facilitate release of the BP cargo in the endosomal environment. RALA/BP nanoparticles were more potent anticancer agents than their free BP counterparts in assays investigating the viability of PC3 prostate cancer and MDA-MB-231 breast cancer cells. Moreover, RALA complexation potentiated the tumor growth delay activity of alendronate in a PC3 xenograft model of prostate cancer. Taken together, these findings further validate the use of BPs as repurposed anticancer agents.

FKBPL is a critical antiangiogenic regulator of developmental and pathological angiogenesis.

OBJECTIVE: The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models. APPROACH AND RESULTS: FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL's critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl(+/-) mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish. CONCLUSIONS: FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.

Peptide delivery of a multivalent mRNA SARS-CoV-2 vaccine.

Lipid nanoparticles (LNP) have been instrumental in the success of mRNA vaccines and have opened up the field to a new wave of therapeutics. However, what is ahead beyond the LNP? The approach herein used a nanoparticle containing a blend of Spike, Membrane and Envelope antigens complexed for the first time with the RALA peptide (RALA-SME). The physicochemical characteristics and functionality of RALA-SME were assessed. With >99% encapsulation, RALA-SME was administered via intradermal injection in vivo, and all three antigen-specific IgG antibodies were highly significant. The IgG2a:IgG1 ratio were all >1.2, indicating a robust TH1 response, and this was further confirmed with the T-Cell response in mice. A complete safety panel of markers from mice were all within normal range, supported by safety data in hamsters. Vaccination of Syrian Golden hamsters with RALA-SME derivatives produced functional antibodies capable of neutralising SARS-CoV-2 from both Wuhan-Hu-1 and Omicron BA.1 lineages after two doses. Antibody levels increased over the study period and provided protection from disease-specific weight loss, with inhibition of viral migration down the respiratory tract. This peptide technology enables the flexibility to interchange and add antigens as required, which is essential for the next generation of adaptable mRNA vaccines.

Combinatorial use of bone morphogenetic protein 6, noggin and SOST significantly predicts cancer progression.

Emerging evidence has indicated a role of the bone morphogenetic proteins (BMP) in the pathogenesis of certain cancers. The signaling of BMP family members is tightly regulated by their antagonists, including noggin and SOST, which are, in turn, positively regulated by BMP, thereby forming a negative feedback loop. Consequently, the expression of these antagonists should be taken into account in studies on the prognostic significance of BMP. In the present paper, we correlated protein and mRNA expression levels of BMP6, noggin and SOST, alone or in combination, with patient survival in various types of cancer. We found that BMP6 alone was not significantly correlated with esophageal squamous cell carcinoma patient survival. Instead, a high level of inhibitor of differentiation 1, a downstream factor of BMP6, was associated with shorter survival in patients whose tumors stained strongly for BMP6. Knockdown of noggin in esophageal cancer cell line EC109, which expresses BMP6 strongly and SOST weakly, enhanced the non-adherent growth of the cells. Noggin and SOST expression levels, when analyzed alone, were not significantly correlated with patient survival. However, high BMP6 activity, defined by strong BMP6 expression coupled with weak noggin or SOST expression, was significantly associated with shorter survival in esophageal squamous cell carcinoma patients. We further confirmed that BMP6 activity could be used as a prognostic indicator in prostate, bladder and colorectal cancers, using publicly available data on BMP6, noggin and SOST mRNA expression and patient survival. Our results strongly suggest that BMP6, noggin and SOST could be used in combination as a prognostic indicator in cancer progression.

Hydrogel-Forming Microneedle Arrays for Enhanced Transdermal Drug Delivery.

Unique microneedle arrays prepared from crosslinked polymers, which contain no drug themselves, are described. They rapidly take up skin interstitial fluid upon skin insertion to form continuous, unblockable, hydrogel conduits from attached patch-type drug reservoirs to the dermal microcirculation. Importantly, such microneedles, which can be fabricated in a wide range of patch sizes and microneedle geometries, can be easily sterilized, resist hole closure while in place, and are removed completely intact from the skin. Delivery of macromolecules is no longer limited to what can be loaded into the microneedles themselves and transdermal drug delivery is now controlled by the crosslink density of the hydrogel system rather than the stratum corneum, while electrically modulated delivery is also a unique feature. This technology has the potential to overcome the limitations of conventional microneedle designs and greatly increase the range of the type of drug that is deliverable transdermally, with ensuing benefits for industry, healthcare providers and, ultimately, patients.

The role of Pea3 group transcription factors in esophageal squamous cell carcinoma.

The transcription factors Pea3, Erm, and Er81 can promote cancer initiation and progression in various types of solid tumors. However, their role in esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we found that the expression levels of Pea3 and Erm, but not that of Er81, were significantly higher in ESCC compared with nontumor esophageal epithelium. A high level of Pea3 expression was significantly correlated with a shorter overall survival in a cohort of 81 patients with ESCC and the subgroup with N1 stage tumor (Wilcoxon-Gehan test, P = 0.016 and P = 0.001, respectively). Pea3 was overexpressed in seven ESCC cell lines compared with two immortalized esophageal cell lines. Pea3 knockdown reduced cell proliferation and suppressed nonadherent growth, migration, and invasion in ESCC cells in vitro. In addition, Pea3 knockdown in ESCC cells resulted in a down-regulation of phospho-Akt and matrix metalloproteinase 13, whereas a significant positive correlation in the expression levels was observed between Pea3 and phospho-Akt (r = 0.281, P < 0.013) and between Pea3 and matrix metalloproteinase 13 in the human specimens (r = 0.462, P < 0.001). Moreover, Pea3 modulated the sensitivity of EC109 cells to doxorubicin, probably via reduced activity of the phosphatidylinositol 3-kinase-Akt-mammalian target of Rapamycin complex 1 pathway on Pea3 knockdown. In conclusion, our results suggest that Pea3 plays an important role in the progression of ESCC.

Polyomavirus enhancer activator 3 protein promotes breast cancer metastatic progression through Snail-induced epithelial-mesenchymal transition.

Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer.

Rational design and characterisation of an amphipathic cell penetrating peptide for non-viral gene delivery.

RALA is a cationic amphipathic peptide which has shown great promise as an efficient, multifunctional delivery system for the delivery of nucleic acids. Rational peptide design was utilised in this study to understand the essential amino acids required for delivery and if any improvements to the RALA peptide could be made. Six amphipathic peptides were synthesised with strategic sequences and amino acid substitutions to reduce peptide sequence, while maintaining the functional characteristics of RALA including amphipathicity, alpha-helicity and pH responsiveness for endosomal escape. Data demonstrated that all six peptides complexed pEGFP-N1 to produce cationic nanoparticles <200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for cellular uptake and endosomal escape. Pep2, produced nanoparticles with similar characteristics and transfection efficiency to the parent peptide, RALA. However, Pep2 had issues with toxicity and a lack of pH-responsive alpha-helcity. Therefore, RALA remains the superior sequence for non-toxic gene delivery.

Correction: TAZ Expression as a Prognostic Indicator in Colorectal Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0054211.].

Rational design and characterisation of a linear cell penetrating peptide for non-viral gene delivery.

The design of a non-viral gene delivery system that can release a functional nucleic acid at the intracellular destination site is an exciting but also challenging proposition. The ideal gene delivery vector must be non-toxic, non-immunogenic, overcome extra- and intra-cellular barriers, protect the nucleic acid cargo from degradation with stability over a range of temperatures. A new 15 amino acid linear peptide termed CHAT was designed in this study with the goal of delivering DNA with high efficiency into cells in vitro and tissues in vivo. Rational design involved incorporation of key amino acids including arginine for nucleic acid complexation and cellular uptake, tryptophan to enhance hydrophobic interaction with cell membranes, histidine to facilitate endosomal escape and cysteine for stability and controlled cargo release. Six linear peptides were synthesised with strategic sequences and amino acid substitutions. Data demonstrated that all six peptides complexed pDNA to produce cationic nanoparticles less than 200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for endosomal escape. Peptide 4, now termed CHAT, was non-cytotoxic, traversed the plasma membrane of breast and prostate cancer cell lines, and elicited reporter-gene expression following intra-tumoural and intravenous delivery in vivo. CHAT presents an exciting new peptide for the delivery of nucleic acid therapeutics.

NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes post-ischaemic revascularization.

AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.

Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide.

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.

DNA vaccination via RALA nanoparticles in a microneedle delivery system induces a potent immune response against the endogenous prostate cancer stem cell antigen.

Castrate resistant prostate cancer (CRPC) remains a major challenge for healthcare professionals. Immunotherapeutic approaches, including DNA vaccination, hold the potential to harness the host's own immune system to mount a cell-mediated, anti-tumour response, capable of clearing disseminated tumour deposits. These anti-cancer vaccines represent a promising strategy for patients with advanced disease, however, to date DNA vaccines have demonstrated limited efficacy in clinical trials, owing to the lack of a suitable DNA delivery system. This study was designed to evaluate the efficacy of a two-tier delivery system incorporating cationic RALA/pDNA nanoparticles (NPs) into a dissolvable microneedle (MN) patch for the purposes of DNA vaccination against prostate cancer. Application of NP-loaded MN patches successfully resulted in endogenous production of the encoded Prostate Stem Cell Antigen (PSCA). Furthermore, immunisation with RALA/pPSCA loaded MNs elicited a tumour-specific immune response against TRAMP-C1 tumours ex vivo. Finally, vaccination with RALA/pPSCA loaded MNs demonstrated anti-tumour activity in both prophylactic and therapeutic prostate cancer models in vivo. This is further evidence that this two-tier MN delivery system is a robust platform for prostate cancer DNA vaccination. STATEMENT OF SIGNIFICANCE: This research describes the development and utilisation of our unique microneedle (MN) DNA delivery system, which enables penetration through the stratum corneum and deposition of the DNA within the highly immunogenic skin layers via a dissolvable MN matrix, and facilitates cellular uptake via complexation of pDNA cargo into nanoparticles (NPs) with the RALA delivery peptide. We report for the first time on using the NP-MN platform to immunise mice with encoded Prostate Stem Cell Antigen (mPSCA) for prostate cancer DNA vaccination. Application of the NP-MN system resulted in local mPSCA expression in vivo. Furthermore, immunisation with the NP-MN system induced a tumour-specific cellular immune response, and inhibited the growth of TRAMP-C1 prostate tumours in both prophylactic and therapeutic challenge models in vivo.

Identification and pharmaceutical evaluation of novel frog skin-derived serine proteinase inhibitor peptide-PE-BBI (Pelophylax esculentus Bowman-Birk inhibitor) for the potential treatment of cancer.

Amphibian venom-derived peptides have high potential in the field of anticancer drug discovery. We have isolated a novel Bowman-Birk proteinase inhibitor (BBI)-type peptide from the skin secretion of Pelophylax esculentus (PE) named PE-BBI, and evaluated its bio-functions and anti-cancer activity in vitro. PE-BBI is a heptadecapeptide with C-terminal amidation. The mRNA sequence and primary structure of PE-BBI were identified using RT-PCR and LC/MS, respectively. A trypsin inhibitory assay was used to characterize the serine proteinase inhibitory activity of synthetic PE-BBI. PE-BBI's myotropic activity was analyzed using isolated rat bladder and rat-tail artery smooth muscle tissues, and the anti-cancer ability of PE-BBI using human colorectal cancer cells. PE-BBI's mechanism of action was investigated using Discovery studio software. PE-BBI showed trypsin inhibitory activity (Ki = 310 ± 72 nM), strong myotropic activity, and cytotoxicity that were specific to cancer cells, and no side effect to normal epithelial cells. The docking stimulation showed that PE-BBI had high affinity to several members of human kallikrein related peptidase (KLK) family. This finding helps to enrich our understanding of BBI peptides' mode of action. Moreover, the data presented here validates frog secretions as sources of potential novel proteinase inhibitors for cancer treatment.

The anti-psychotic drug pimozide is a novel chemotherapeutic for breast cancer.

Pimozide, an antipsychotic drug of the diphenylbutylpiperidine class, has been shown to suppress cell growth of breast cancer cells in vitro. In this study we further explore the inhibitory effects of this molecule in cancer cells. We found that Pimozide inhibited cell proliferation in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells and A549 lung cancer cells. Furthermore, we found that Pimozide also promoted apoptosis as demonstrated by cell cycle arrest and induction of double-strand DNA breaks but did not result in any effect in the non-transformed MCF10A breast cell line. In order to shed new lights into the molecular pathways affected by Pimozide, we show that Pimozide downregulated RAN GTPase and AKT at both protein and mRNA levels and inhibited the AKT signaling pathway in MDA-MB-231 breast cancer cells. Pimozide also inhibited the epithelial mesenchymal transition and cell migration and downregulated the expression of MMPs. Administration of Pimozide showed a potent in vivo antitumor activity in MDA-MB-231 xenograft animal model and reduced the number of lung metastases by blocking vascular endothelial growth factor receptor 2. Furthermore, Pimozide inhibited myofibroblast formation as evaluated by the reduction in α-smooth muscle actin containing cells. Thus, Pimozide might inhibit tumor development by suppressing angiogenesis and by paracrine stimulation provided by host reactive stromal cells. These results demonstrate a novel in vitro and in vivo antitumor activity of Pimozide against breast and lung cancer cells and provide the proof of concept for a putative Pimozide as a novel approach for cancer therapy.

DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system.

Dissolvable microneedles can be employed to deliver DNA to antigen presenting cells within the skin. However, this technology faces two main challenges: the poor transfection efficacy of pDNA following release from the microneedle matrix, and the limited loading capacity of the micron-scale devices. Two-tier delivery systems combining microneedle platforms and DNA delivery vectors have increased efficacy but the challenge of increasing the loading capacity remains. This study utilised lyophilisation to increase the loading of RALA/pDNA nanoparticles within dissolvable PVA microneedles. As a result, delivery was significantly enhanced in vivo into an appropriate range for DNA vaccination (∼50 µg per array). Furthermore, modifying the manufacturing process was not detrimental to the microneedle mechanical properties or cargo functionality. It was demonstrated that arrays retained mechanical and functional stability over short term storage, and were able to elicit gene expression in vitro and in vivo. Finally, treatment with this novel formulation significantly retarded the growth of established tumours, and proved superior to standard intramuscular injection in a preclinical model of cervical cancer.

Gene therapy with RALA/iNOS composite nanoparticles significantly enhances survival in a model of metastatic prostate cancer.

BACKGROUND: Recent approvals of gene therapies by the FDA and the EMA for treatment of inherited disorders have further opened the door for assessment of nucleic acid pharmaceuticals for clinical usage. Arising from the presence of damaged or inappropriate DNA, cancer is a condition particularly suitable for genetic intervention. The RALA peptide has been shown to be a potent non-viral delivery platform for nucleic acids. This study examines the use of RALA to deliver a plasmid encoding inducible nitric oxide synthase (iNOS) as an anti-cancer treatment. METHODS: The physiochemical properties of the RALA/DNA nanoparticles were characterized via dynamic light scattering and transmission electron microscopy. The nanoparticles were labelled with fluorophores and tracked over time using confocal microscopy with orthogonal sections to determine cellular location. In vitro studies were employed to determine functionality of the nanoparticles both for pEGFP-N1 and CMV-iNOS. Nanoparticles were injected intravenously into C57/BL6 mice with blood and serum samples analysed for immune response. PC3-luc2M cells were injected into the left ventricle of SCID mice followed by treatment with RALA/CMV-iNOS nanoparticles to evaluate the tumour response in a metastatic model of prostate cancer. RESULTS: Functional cationic nanoparticles were produced with gene expression in PC-3 prostate cancer cells. Furthermore, repeated administrations of RALA/DNA nanoparticles into immunocompetent mice did not produce any immunological response: neutralization of the vector or release of inflammatory mediators. RALA/CMV-iNOS reduced the clonogenicity of PC-3 cells in vitro, and in an in vivo model of prostate cancer metastasis, systemically delivered RALA/CMV-iNOS significantly improved the survival of mice. CONCLUSION: These studies further validate RALA as a genetic cargo delivery vehicle and iNOS as a potent therapy for the treatment of cancer.

The prognostic significance of Cdc6 and Cdt1 in breast cancer.

DNA replication is a critical step in cell proliferation. Overexpression of MCM2-7 genes correlated with poor prognosis in breast cancer patients. However, the roles of Cdc6 and Cdt1, which work with MCMs to regulate DNA replication, in breast cancers are largely unknown. In the present study, we have shown that the expression levels of Cdc6 and Cdt1 were both significantly correlated with an increasing number of MCM2-7 genes overexpression. Both Cdc6 and Cdt1, when expressed in a high level, alone or in combination, were significantly associated with poorer survival in the breast cancer patient cohort (n = 1441). In line with this finding, the expression of Cdc6 and Cdt1 was upregulated in breast cancer cells compared to normal breast epithelial cells. Expression of Cdc6 and Cdt1 was significantly higher in ER negative breast cancer, and was suppressed when ER signalling was inhibited either by tamoxifen in vitro or letrozole in human subjects. Importantly, breast cancer patients who responded to letrozole expressed significantly lower Cdc6 than those patients who did not respond. Our results suggest that Cdc6 is a potential prognostic marker and therapeutic target in breast cancer patients.