TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells.

Cancer cells often acquire a constitutively active nuclear factor-kappaB (NF-kappaB) program to promote survival, proliferation and metastatic potential by mechanisms that remain largely unknown. Extending observations from an immunologic setting, we demonstrate that microRNA-146a and microRNA-146b (miR-146a/b) when expressed in the highly metastatic human breast cancer cell line MDA-MB-231 function to negatively regulate NF-kappaB activity. Lentiviral-mediated expression of miR-146a/b significantly downregulated interleukin (IL)-1 receptor-associated kinase and TNF receptor-associated factor 6, two key adaptor/scaffold proteins in the IL-1 and Toll-like receptor signaling pathway, known to positively regulate NF-kappaB activity. Impaired NF-kappaB activity was evident from reduced phosphorylation of the NF-kappaB inhibitor IkappaBalpha, reduced NF-kappaB DNA-binding activity and suppressed expression of the NF-kappaB target genes IL-8, IL-6 and matrix metalloproteinase-9. Functionally, miR-146a/b-expressing MDA-MB-231 cells showed markedly impaired invasion and migration capacity relative to control cells. These findings implicate miR-146a/b as a negative regulator of constitutive NF-kappaB activity in a breast cancer setting and suggest that modulating miR-146a/b levels has therapeutic potential to suppress breast cancer metastases.

Increased target specificity of anti-HER2 genospheres by modification of surface charge and degree of PEGylation.

Genospheres are cationic lipid-nucleic acid nanoparticles prepared by the assembly of the lipids and nucleic acids from an aqueous/organic liquid monophase that independently dissolves the components, where the resultant particles are homogeneously sized (70-110 nm), with efficiently incorporated and protected DNA. In the present study, we demonstrate pH-dependent modulation of the Genosphere surface charge using pH-titratable lipids. By incorporation of the lipids with titratable anionic or imidazole headgroups, Genospheres with neutral or anionic surface charge at neutral pH were produced and compared for cellular uptake and transfection of a reporter gene (luciferase) in culture of breast cancer cells. The extent of particle-cell association was also studied by fluorescent microscopy and quantified by cytofluorometery. The effects of Genosphere surface modification with poly(ethylene glycol) (molecular weight 2000) at low (0.5 mol %) and high (5 mol %) grafting densities, as well as the effects of HER2-receptor-directed targeting by an internalizable anti-HER2 scFv F5, linked via PEG spacer, were also studied. Inclusion in the Genosphere formulation of pH-titratable lipids CHEMS (cholesteryl hemisuccinate), CHIM (1-(3-(cholesteryloxycarbonylamino)propyl)imidazole), or DSGG (1,2-distearoyl-sn-glycero-3-hemiglutarate) rendered the particles surface-charge neutral or slightly anionic at neutral pH, and cationic at mildly acidic pH, as shown by zeta-potential measurements. In HER2-targeted systems, transfection activity and target specificity with HER2-overexpressing SKBR-3 breast cancer cells were dependent on Genosphere surface charge and PEGylation. The highest target specificity correlated with low cationic charge at neutral pH, while incorporation of 5 mol % PEG-lipid had only minor effects on Genosphere-cell association, internalization, and transfection activity. The implications of this work for potential in vivo applications are discussed.

Genospheres: self-assembling nucleic acid-lipid nanoparticles suitable for targeted gene delivery.

We describe the assembly of a cationic lipid-nucleic acid nanoparticle from a liquid monophase containing water and a water miscible organic solvent where both lipid and DNA components are separately soluble prior to their combination. Upon removal of the organic solvent, stable and homogenously sized (70-100 nm) lipid-nucleic acid nanoparticles (Genospheres) were formed. The low accessibility (<15%) of the nanoparticle-encapsulated DNA to a DNA intercalating dye indicated well-protected nucleic acids and high DNA incorporation efficiencies. It was demonstrated that Genospheres could be stably stored under a variety of conditions including a lyophilized state where no appreciable increase in particle size or DNA accessibility was observed following reconstitution. Finally, Genospheres were made target-specific by insertion of an antibody-lipopolymer (anti-HER2 scFv (F5)-PEG-DSPE) conjugate into the particle. The target specificity (>100-fold) in HER2 overexpressing SK-BR-3 breast cancer cells was dependent on the degree of PEGylation, where the incorporation of high amounts of PEG-lipid on the particle surface (up to 5 mol%) had only a minor effect on the transfection activity of the targeted Genospheres. In summary, this work describes a novel, readily scalable method for preparing highly stable immunotargeted nucleic acid delivery vehicles capable of achieving a high degree of specific transfection activity.

Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment.

Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice.

The NFkappaB pathway and endocrine-resistant breast cancer.

Endocrine therapy with an estrogen receptor (ER)-targeted antiestrogen, such as tamoxifen, or estrogen ablation by aromatase inhibitors is clinically indicated for the management of all forms of ER-positive breast cancer. However, 30-50% of ER-positive breast cancer cases fail to benefit clinically from endocrine therapy alone, and recent molecular evidence suggests that 'crosstalk' pathways originating from activated receptor tyrosine kinases and/or other proliferative and survival signals may be contributing to this endocrine resistance. Molecular identification and validation of candidate ER crosstalking pathways will likely lead to clinically important prognostic markers and targets for the application of novel therapeutics in combination with standard endocrine agents. This review focuses on a critical survival and proliferation pathway involving activation of nuclear factor-kappaB (NFkappaB), a family of ubiquitously expressed transcription factors that for nearly two decades have been known to be critical regulators of mammalian immune and inflammatory responses, and more recently have been associated with chemotherapy resistance. With the demonstration that activation of NFkappaB is absolutely required for normal mammary gland development, NFkappaB involvment in human breast cancers was initially explored and linked to the development of hormone-independent (ER-negative) breast cancer. Newer clinical evidence now implicates NFkappaB activation, particularly DNA-binding by the p50 subunit of NFkappaB, as a potential prognostic marker capable of identifying a high-risk subset of ER-positive, primary breast cancers destined for early relapse despite adjuvant endocrine therapy with tamoxifen. Furthermore, initial preclinical studies suggest that treatment strategies designed to prevent or interrupt activation of NFkappaB in cell-line models of these more aggressive, ER-positive breast cancers can restore their sensitivity to such standard endocrine agents as tamoxifen.

Understanding the age dependency of breast cancer biomarkers.

This review and study of nearly 4,000 primary breast cancers evaluates the hypothesis that human aging not only increases breast cancer incidence but also alters breast cancer biology. Clinically validated biomarkers were chosen as surrogate measures of genetic instability and tumor growth, invasiveness and metastatic potential. Our results support the premise that breast cancer clinical behavior and biology are significantly affected by patient age, but call into question key aspects of the current cancer-aging paradigm.

Tumor targeting using anti-her2 immunoliposomes.

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.

Expression of a truncated 100 kDa HER2 splice variant acts as an endogenous inhibitor of tumour cell proliferation.

Overexpression of the HER2 (neu/c-erbB-2) oncogene frequently coincides with an aggressive clinical course of certain human adenocarcinomas. Expression and secretion of aberrant HER2 splice variants has been reported in various cell lines and tissues and can interfere with the oncogenic HER2 activity. Here we demonstrate, using two different approaches, that expression of a truncated 100 kDa HER2 variant which encodes the extracellular domain of HER2 (HER-ECD) inhibits growth factor-mediated tumour cell proliferation. A HER2-ECD cDNA encoding the truncated variant was overexpressed in MCF7 breast cancer cells. HER2-ECD overexpression decreased spontaneous proliferation of MCF7 cells as well as heregulin-mediated soft agar colony formation. Concomitantly, heregulin-induced phosphorylation of HER4 as well as downstream activation of p44/p42 MAP-kinases was decreased. To confirm these data, ribozymes were targeted to the 3'-untranslated region of the 2.3 kb HER2-ECD mRNA which is spontaneously expressed in MKN7 gastric cancer cells. HER2-ECD-targeted ribozymes downregulated HER2-ECD expression and enhanced EGF-mediated soft agar colony formation of MKN7 cells. In parallel, EGF-induced activation of p44/p42 MAP-kinases and activation of c-Fos expression were increased in ribozyme-transfected MKN7 cells. Finally, in RT-PCR we found a trend towards a progressive loss of 2.3 kb HER2-ECD mRNA expression in more advanced gastric tumours. These data show that the HER2-ECD variant inhibits growth factor-mediated tumour cell proliferation suggesting an important role during the progression of human cancer.

Biological effects of anti-ErbB2 single chain antibodies selected for internalizing function.

Two internalizing monovalent single chain antibody fragments (scFv), C6.5 and F5, that recognize distinct ErbB2 extracellular domain (ECD) epitopes, and their bivalent forms dbC6.5 and F5(scFv')(2), were compared to the growth-inhibiting anti-ErbB2 antibody Herceptin/trastuzumab, in either its bivalent (Her) or monovalent (4D5Fab') form, for their abilities to induce biological responses in the ErbB2-overexpressing breast cancer cells, SkBr-3. Assays compared internalization by receptor-mediated endocytosis, effects on cell cycling and culture growth, and interference with intracellular MAPK and PI3K signaling pathways. We found no correlation between ErbB2 epitope affinity or valency on degree of antibody-induced endocytosis, since all the scFv were able to internalize better than Her. Unlike Her, neither the monovalent or bivalent forms of the internalizing scFv had any sustained effect on cell growth. Basal levels of MAPK and PI3K signaling in SkBr-3 cells were not inhibited by up to 8 h scFv treatment, while decreased MAPK and PI3K signals were noted within 8 h of Her treatment. In summary, antibody-induced ErbB2-mediated endocytosis is not a surrogate marker for resultant biological response, as it shows no correlation with cell cycle, culture proliferation, or intracellular kinase signal induction by internalizing antibodies. Thus, the enhanced endocytotic property of scFv like C6.6 and F5 in conjunction with their absence of any growth or signaling impact on ErbB2-overexpressing cells favors their choice as ErbB2 targeting moieties for intracellular delivery of novel cancer therapeutics.

Liposome targeting to tumors using vitamin and growth factor receptors.

Liposome-encapsulated anticancer drugs reveal their potential for increased therapeutic efficacy and decreased nonspecific toxicities due to their ability to enhance the delivery of chemotherapeutic agents to solid tumors. Advances in liposome technology have resulted in the development of ligand-targeted liposomes capable of selectively increasing the efficacy of carried agents against receptor-bearing tumor cells. Receptors for vitamins and growth factors have become attractive targets for ligand-directed liposomal therapies due to their high expression levels on various forms of cancer and their ability to internalize after binding to the liposomes conjugated to receptors' natural ligands (vitamins) or synthetic agonists (receptor-specific antibodies and synthetic peptides). This chapter summarizes various strategies and advances in targeting liposomes to vitamin and growth factor receptors in vitro and in vivo with special emphasis on two extensively studied liposome-targeting systems utilizing folate receptor and HER2/neu growth factor receptor.

Activation (tyrosine phosphorylation) of ErbB-2 (HER-2/neu): a study of incidence and correlation with outcome in breast cancer.

PURPOSE: We hypothesize that phosphorylated ErbB-2 (P-ErbB-2, identified by a novel antibody PN2A) may provide either more significant or additional prognostic marker data for breast cancer patients. This study was designed to compare the incidence and prognostic value of ErbB-2 (HER-2/neu) and P-ErbB-2 immunoexpression in archival breast cancer samples. MATERIALS AND METHODS: Eight hundred sixteen invasive breast cancers with a median of 16.3 years of follow-up were immunostained for ErbB-2 (using antibody CB11) and P-ErbB-2 (using antibody PN2A). ErbB-2 and P-ErbB-2 data were compared with clinical, histologic, immunohistochemical, and outcome variables. RESULTS: Of 816 primary breast cancers, 307 (38%) were positive for ErbB-2 and 37 (12% of ErbB-2 positive and 5% of the study population) expressed P-ErbB-2. P-ErbB-2 was not detected in ErbB-2-negative cases (n = 509). ErbB-2 immunohistochemical data were bimodal; patients with > or = 80% cellular expression had the shortest disease-free and disease-specific survival. P-ErbB-2 was associated with a higher percentage of ErbB-2-positive cells, a higher number of positive lymph nodes, and cellular proliferation. ErbB-2 and P-ErbB-2 were indicators of poor prognosis in node-positive patients in both univariate and multivariate analyses. We found that either P-ErbB-2 expression or high (> or = 80%) ErbB-2 expression provided the most significant prognostic value in node-positive cases by multivariate analyses. There were too few P-ErbB-2-positive cases and events in the node-negative patient group to allow statistical analysis of P-ErbB-2 in that subgroup. CONCLUSION: PN2A immunostaining identified a subset (approximately 12% of ErbB-2-positive breast cancers) with activation (phosphorylation) of the receptor ErbB-2. P-ErbB-2 expression was strongly associated with higher levels of ErbB-2 expression (> or = 80%), although it was not a surrogate. Identification of cases with a high percentage of invasive breast cancer cells expressing ErbB-2 or determination of receptor activation via P-ErbB-2 may provide additional prognostic value in node-positive breast cancers.

Preferential oxidation of zinc finger 2 in estrogen receptor DNA-binding domain prevents dimerization and, hence, DNA binding.

For approximately one-third of estrogen receptor (ER)-positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiol-reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide; however, prior DNA binding by ER or ER-DBD protects against this oxidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognition helix and a flexible helix-supported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ER-DBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ER-DBD structure (alpha-helicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-binding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNA-binding function in extracts from some primary breast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ER-DBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.

Tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents.

Abnormal cell signal transduction arising from protein tyrosine kinases has been implicated in the initiation and progression of a variety of human cancers. Over the past 2 decades pharmaceutical and university laboratories have been involved in a tremendous effort to develop compounds that can selectively modulate these abnormal signalling pathways. Targeting receptor tyrosine kinases, especially the epidermal growth factor receptor subfamily, has been at the forefront of this effort as a result of strong clinical data correlating over-expression of these receptors with more aggressive cancers. There are a variety of strategies under development for inhibiting the kinase activity of these receptors, targeting both the extracellular and intracellular domains. Antibody-based approaches, immunotoxins and ligand-binding cytotoxic agents use the extracellular domain for targeted tumour therapy. Small molecule inhibitors target the intracellular catalytic region by interfering with ATP binding, while nonphosphorylatable peptides are aimed at the intracellular substrate binding region. Compounds that inhibit subsequent downstream signals from the receptor by interrupting intracellular protein recognition sequences are also being investigated. In the past 5 years enormous progress has been made in developing tyrosine kinase inhibitor compounds with sufficient potency, bioavailability and selectivity against this subfamily of receptor tyrosine kinases. The anti-HER2 monoclonal antibody, trastuzumab, for patients with metastatic breast cancer is the first of these inhibitor compounds to gain FDA approval. However, preclinical and clinical trials are ongoing with a variety of other monoclonal antibodies, immunotoxins, and small molecule quinazoline and pyrimidine-based inhibitors. Although their cytotoxic and cytostatic potential has been proven, they are not likely to replace standard chemotherapy regimens as single-agent, first-line therapeutics. Instead, their promising additive and synergistic antitumour effects in combination with standard chemotherapeutics suggest that these novel agents will find their greatest utility and efficacy in conjunction with existing anticancer agents.

Targeting the ets binding site of the HER2/neu promoter with pyrrole-imidazole polyamides.

Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(-1) to 1.4. 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.

Ets regulation of the erbB2 promoter.

Evaluating the chromatinized erbB2 gene in nuclei from breast cancer cells expressing varying levels of ErbB2 transcripts, we identified a nuclease-sensitive site within a 0.22 kb region of maximum enhancer activity centered over a conserved 28 bp polypurine(GGA)-polypyrimidine(TCC) mirror-repeat and an adjacent essential Ets binding site (EBS). Promoter footprinting with nuclear extracts reveals an intense Ets hypersensitivity site at the EBS whose degree of intensity correlates with the level of cellular ErbB2 expression. In vitro mapping assays show that the supercoiled erbB2 promoter forms an internal triplex structure (Hr-DNA) at the mirror-repeat element. Mutations preventing Hr-DNA formation can enhance erbB2 promoter activity in human breast cancer cells, a result consistent with previous demonstration that Ets-erbB2 promoter complexes cannot form when the mirror-repeat is engaged in triplex binding, and new results suggesting that Ets binding induces severe promoter bending that may restrict local triplex formation. In addition to previously described erbB2-regulating breast cancer Ets factors (PEA3, ESX/Elf-3), Elf-1 is now shown to be another endogenously expressed Ets candidate capable of binding to and upregulating the erbB2 promoter. Given current strategies to transcriptionally inhibit ErbB2 overexpression, including development of novel erbB2 promoter-targeted therapeutics, an EBS-targeted approach is presented using chimeric Ets proteins that strongly repress erbB2 promoter activity.

Emerging antibody-based HER2 (ErbB-2/neu) therapeutics.

Targeting HER2(ErbB-2/neu) overexpressing tumor cells to selectively deliver anticancer agents and thereby reduce host toxicity represents a rational and emerging strategy for the treatment of breast and other epithelial cancers. The extracellular domain of the HER2 receptor tyrosine kinase is readily accessible to systemically administered antibody-based therapeutics, including growth-inhibiting monclonals such as rhuMAbHER2 (trastuzmab/Herceptin) as well as anti-HER2 immunotoxins, antibody-dependent enzyme prodrug therapy (ADEPT), and immune cell recruiting bispecific antibodies. In addition to summarizing recent advances in these antibody-based strategies, this review focuses on preclinical advances in the development of anti-HER2 immunoliposomes (ILs) as a platform technology for targeted drug delivery. Extensive in vitro and in vivo testing including efficacy and tumor uptake studies in multiple human tumor xenograft models now provide conclusive evidence for the superior therapeutic efficacy of anti-HER2 ILs-doxorubicin (dox) over free dox or liposomal (Ls)-dox, and even over combinations of dox and Ls-dox with rhuMAbHER2. As anti-HER2 ILs-dox approaches clinical testing in patients with advanced HER2 overexpressing breast cancer, future applications of this novel targeting strategy will also broaden to include intracellular delivery of other anticancer agents as well as therapeutic nucleic acids (oligonucleotides, genes).

Constitutive expression of Heregulin induces apoptosis in an erbB-2 overexpressing breast cancer cell line SKBr-3.

We have previously reported that Heregulin (HRG)/neu differentiation factor (NDF) induced growth arrest and cellular differentiation in breast cancer cells overexpressing erbB-2 receptor. To elucidate the cellular mechanisms underlying the growth inhibition by HRG, we developed an in vitro model by transfection of HRG cDNA into the erbB-2 overexpressing breast cancer cell line, SKBr-3. We showed that the enforced expression of HRG in SKBr-3 cells induces dramatic morphological changes and pronounced inhibition of anchorage-dependent and -independent growth. Most SKBr-3/HRG-transfected (SK/HRG) cells exhibited about 15-fold increase in size and persisted as multinucleated cells with extended flattened vacuole-filled cytoplasm with reduced cell attachment. The growth suppression of SK/HRG cells was accompanied by a reduction in S phase, the presence of a G2-M cell cycle delay, and an increase in DNA aneuploid components. In addition, DNA fragmentation assays showed that HRG induced apoptosis of SKBr-3 cells. In contrast, while HRG treatment of other erbB-2 overexpressing breast cancer cell lines led to growth arrest and cell detachment, it did not induce apoptotic features. Thus, this study demonstrates that while growth arrest and cell detachment are general effects of HRG towards erbB-2 overexpressing cells, the ability of HRG to induce apoptosis is a phenomenon confined to selective cells including SKBr-3 cells.