Discovery and Pharmacological Evaluation of STEAP4 as a Novel Target for HER2 Overexpressing Breast Cancer.

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

Targeting of the breast cancer microenvironment with a potent and linkable oxindole based antiangiogenic small molecule.

The clinical efficacy of antiangiogenic small molecules (e.g., sunitinib) in breast carcinoma has largely failed with substantial off-target toxicity. We rationally designed and evaluated preclinically a novel sunitinib analogue, SAP, with favourable pharmacological properties and the ability to be readily conjugated to a targeting peptide or antibody for active tumour targeting.SAP was evaluated in silico and in vitro in order to verify target engagement (e.g., VEGFR2). Pharmacokinetic and biodistribution parameters were determined in mice using LC-MS/MS. SAP efficacy was tested in two breast cancer xenograft and two syngeneic animal models and pharmacodynamic evaluation was accomplished using phosphokinase assays and immunohistochemistry. Cardiac and blood toxicity of SAP were also monitored.SAP retained the antiangiogenic and cytotoxic properties of the parental molecule with an increased blood exposure and tumor accumulation compared to sunitinib. SAP proved efficacious in all animal models. Tumors from SAP treated animals had significantly decreased Ki-67 and CD31 markers and reduced levels of phosphorylated AKT, ERK and S6 compared to vehicle treated animals. In mice dosed with SAP there was negligible hematotoxicity, while cardiac function measurements showed a reduction in the percentage left ventricular fractional shortening compared to vehicle treated animals.In conclusion, SAP is a novel rationally designed conjugatable small antiangiogenic molecule, efficacious in preclinical models of breast cancer.

Gemcitabine Based Peptide Conjugate with Improved Metabolic Properties and Dual Mode of Efficacy.

Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabolically superior to gemcitabine molecule (GSG) by conjugating gemcitabine to a gonadotropin releasing hormone receptor (GnRH-R) ligand peptide and showed that GSG was efficacious in a castration resistant prostate cancer (CRPC) animal model. The current article provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic. LC-MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake, and release of gemcitabine and to quantitate the intracellular levels of gemcitabine and its metabolites (active dFdCTP and inactive dFdU) resulting from GSG. The GnRH-R agonistic potential of GSG was investigated by quantifying the testosterone levels in animals dosed daily with GSG, while an in vitro colony forming assay together with in vivo whole blood measurements were performed to elucidate the hematotoxicity profile of GSG. Stability showed that the major metabolite of GSG is a more stable nonapeptide that could prolong gemcitabine's bioavailability. GSG acted as a prodrug and offered a metabolic advantage compared to gemcitabine by generating higher and steadier levels of dFdCTP/dFdU ratio, while intracellular release of gemcitabine from GSG in DU145 CRPC cells depended on nucleoside transporters. Daily administrations in mice showed that GSG is a potent GnRH-R agonist that can also cause testosterone ablation without any observed hematotoxicity. In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single nontoxic molecule that can be used to reach the tumor site selectively with superior to gemcitabine metabolism, biodistribution, and safety while also agonistically ablating testosterone levels.

Discovery of New Aminosubstituted Pyrrolopyrimidines with Antiproliferative Activity Against Breast Cancer Cells and Investigation of their Effect Towards the PI3Kα Enzyme.

OBJECTIVE: A series of novel 2,4-diaminosubstituted pyrrolo[3,2-d]pyrimidines was synthesized together with their corresponding 7-phenyl or 7-isopropyl counterparts. RESULTS: Among the target derivatives, the 7-substituted analogues exhibited interesting cytotoxic activity against a panel of PI3Kα related human breast cancer cell lines, namely MCF7, T47D, MDA-MB-231 and HCC1954. Selected compounds were tested for potential PI3Kα inhibitory activity as well as for their cytotoxic effect in prostate cancer cell lines (DU145 and PC3). CONCLUSION: Derivatives bearing a specific substitution pattern consisting of 7-phenyl as well as a 2-(4- aminocyclohexylamino) moiety (16c, 16f) display kinase inhibitory activity, elucidated on the basis of molecular simulation studies, which revealed their interaction with the DFG motif of the kinase.

Design and synthesis of novel 7-aminosubstituted pyrido[2,3-b]pyrazines exhibiting anti-breast cancer activity.

Breast cancer (BrCa) remains an unmet medical need despite the revolutionary development of antibody treatments and protein kinase inhibitors. In the current study, a series of novel substituted pyridopyrazine derivatives have been rationally designed and evaluated as multi-kinase inhibitors in the PI3K pathway. The target compounds were prepared from 6-amino-2-picoline, which upon nitration and selective reduction was converted to suitably substituted 6-methyl-7-aminopyrido[2,3-b]pyrazines. Suitable manipulation of the former amines provided the designed analogues, which were then assessed in vitro against several BrCa cell lines using the MTT cytotoxicity assay. The most potent compounds underwent evaluation in a broad spectrum of protein kinases, while their pharmacokinetic parameters were measured by LC-MS/MS. In vivo evaluation of a hit compound (14a) was performed in a HER2 amplified BrCa xenograft model (HCC1954) and efficacy was determined using Western blot based phosphokinase assays and immunohistochemistry. This derivative showed low micromolar cytotoxic potency in all BrCa cell lines, a mild inhibition of the PI3Kα wild type and H1047R mutated enzyme and excellent pharmacokinetic parameters following oral and intraperitoneal administration at the designed dose of 10 mg/kg, with absence of in vivo phenotypic toxicity. Interestingly, compound 14a inhibited the growth of xenografted tumors. Analysis of excised tumors from the treated animals showed a significantly reduced population of Ki-67 positive cells, as well as downregulated levels of phosphorylated AKT, ERK1/2 and SRC compared to vehicle treated animals. Finally, the specificity of 14a was assessed in a panel of 31 kinases where a mild, but direct, inhibition of the MET receptor tyrosine kinase was observed.

Peptide-Drug Conjugate GnRH-Sunitinib Targets Angiogenesis Selectively at the Site of Action to Inhibit Tumor Growth.

The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [d-Lys(6)]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R-expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphorylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide-drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site (∼4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation.

Sustained expression from DNA vectors.

DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.

GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone-Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake, and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely, GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG showed a significant advantage in tumor growth inhibition when compared to gemcitabine.

Examination of the effect of increasing the number of intra-disulfide amino functional groups on the performance of small molecule cyclic polyamine disulfide vectors.

Establishing structure-activity relationships is vital if the efficacy of non-viral vectors is to match that of their viral counter-parts. Recently, we reported on the ability of a series of small molecule, cyclic polyamine disulfides to condense and cage plasmid DNA (pDNA) by a process of thermodynamically controlled templated polymerization, leading to a series of corresponding pDNA-polyplex nanoparticles able to mediate high levels of transfection with no associated cytotoxicities. The leading cyclic polyamine disulfide was shown to be the spermine tetra-amine disulfide (TetraN-3,4,3). Herein we report on the significantly more challenging syntheses of cyclic disulfides with longer polyamine motifs. Two new cyclic polyamine disulfides, based on hexa- and octa-amine inserts, were prepared and their transfection efficacies and cytotoxicities compared with our previously reported cyclic tri- and tetra-amine disulfides. The new cyclic hexa- and octa-amine disulfides prove more effective at transfection in vitro, especially of lung epithelial A549 cell line. By contrast, our original cyclic tetra-amine disulfide remains the most efficient agent for the transfection of lung epithelial cells in vivo following intra-nasal administration. Hypothetical mechanistic reasons are presented to explain this outcome. Our data in toto support the concept of shorter cyclic polyamine disulfides as preferred agents for polycation-mediated controlled condensation and functional delivery of pDNA to lung epithelial cells in vivo.

Genetic modification of cancer cells using non-viral, episomal S/MAR vectors for in vivo tumour modelling.

The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into "immune deficient" mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome.

Vector systems for prenatal gene therapy: principles of non-viral vector design and production.

Gene therapy vectors based on viruses are the most effective gene delivery systems in use today and although efficient at gene transfer their potential toxicity (Hacein-Bey-Abina et al., Science 302:415-419, 2003) provides impetus for the development of safer non-viral alternatives. An ideal vector for human gene therapy should deliver sustainable therapeutic levels of gene expression without affecting the viability of the host at either the cellular or somatic level. Vectors, which comprise entirely human elements, may provide the most suitable method of achieving this. Non-viral vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production, and great versatility. The development of more efficient, economically prepared, and safer gene delivery vectors is a crucial prerequisite for their successful clinical application and remains a primary strategic task of gene therapy research.

Non-viral episomal modification of cells using S/MAR elements.

INTRODUCTION: The early potential of gene therapy is slowly becoming realized following the recent treatment of patients with severe combined immunodeficiency and ocular diseases. However at present the field of gene therapy is tempered by the toxicity issues, mainly that of the integrated retroviral vector used in most trials which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors is therefore vital for further progress in this field, in particular vectors which remain episomal and are therefore less genotoxic. One such unique class of vectors are those based on scaffold matrix attachment regions (S/MARs) elements, which are maintained extra-chromosomally and replicate in vitro and in vivo. AREAS COVERED: The overview here describes the most relevant studies utilizing the S/MAR element to episomally modify mammalian cells and tissues with a particular focus on liver tissue, as well as the brain, the muscle, the eye, cancer cells, embryonic cells and neonatal mice. For this purpose, recently published data in these areas (mainly articles published between 2000 and 2010) are reviewed. EXPERT OPINION: The utilisation of vectors harbouring an S/MAR element is an efficient, safe and cost-effective way to episomally modify mammalian cells.

Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver.

We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.

Systemic gene transfer of polyethylenimine (PEI)-plasmid DNA complexes to neonatal mice.

Non-viral vectors have not been extensively investigated in neonatal mice due to the poor efficiency of the delivery methods available. Understanding the effects of non-viral vectors during early development is vital to develop safe gene therapy treatments where irreversible pathological processes may be avoided by early gene reconstitution. Here we describe a simple and effective method for the systemic administration of non-viral vectors via the superior temporal vein of mouse pups at 1.5 days of age. We show that injection of polyethylenimine (PEI)-complexed plasmid DNA (pDNA) intravenously results in effective transfection in the liver, lung, heart, spleen, brain and kidney. We also investigate the specific targeting of transgene expression to the proliferating neonate liver using a liver-specific plasmid containing a Scaffold Matrix Attachment Region (S/MAR) element, which has previously been shown to confer long-term expression in adult mouse liver. Using bioluminescent imaging, a gradual increase in transgene expression was observed which peaked at days 11-12, before the reduction of expression to background levels by day 25, suggestive of vector copy number loss. We conclude that non-viral vectors can successfully be used for systemic delivery to neonatal mice and that this technique is likely to open a host of early therapeutic possibilities for gene transfer by a range of non-viral vector formulations.

Bioresponsive small molecule polyamines as noncytotoxic alternative to polyethylenimine.

Nonviral gene therapy continues to require novel synthetic vectors to deliver therapeutic nucleic acids effectively and safely. The majority of synthetic nonviral vectors employed in clinical trials to date have been cationic liposomes; however, cationic polymers are attracting increasing attention. One of the few cationic polymers to enter clinical trials has been polyethylenimine (PEI); however, doubts remain over its cytotoxicity, and in addition it displays lower levels of transfection than viral systems. Herein, we report on the development of a series of small molecule analogues of PEI that are bioresponsive to the presence of pDNA, forming poly(disulfide)s that are capable of efficacious transfection with no associated toxicity. The most effective small molecule developed, a cyclic disulfide based upon a spermine backbone, is shown to form very well-defined polyplexes (100-200 nm in diameter) that mediate murine lung transfection in vivo to within an order of magnitude of in vivo jetPEI, and at the same time display a much improved cytotoxicity profile.

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells.

BACKGROUND: The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP) and directed into a 2000 bp long matrix attachment region sequence (MARS) derived from the human beta-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression. RESULTS: Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P) element that is known to be less affected by epigenetic silencing events. CONCLUSIONS: The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

Strategies for the episomal modification of cells.

The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This review describes the development and application of gene therapy vectors, with a focus on those that are specifically designed to avoid integration and exist episomally.