ErbB2 trafficking and degradation associated with K48 and K63 polyubiquitination.

The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185-kDa ErbB2 receptor is rapidly (2-6 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341-treated SK-BR-3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase c-Cbl and also with the deubiquitinating enzyme USP9x; moreover, siRNA downregulation of USP9x enhances PS341-induced ErbB2 downregulation.

Destabilization of ERBB2 transcripts by targeting 3' untranslated region messenger RNA associated HuR and histone deacetylase-6.

In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA.

Proteasome-regulated ERBB2 and estrogen receptor pathways in breast cancer.

A major challenge to broadening oncology applications for inhibitors of the ubiquitin-proteasome system (UPS) is the identification of UPS-dependent cancer pathways predictive of tumors responsive to peptidomimetic inhibitors of its 20S core protease activity. To inform clinical studies evaluating UPS inhibitors as breast cancer therapeutics, seven phenotypically diverse human breast cancer cell line models were characterized for their cellular and molecular responses to the clinically approved 20S inhibitor bortezomib (PS341; Velcade), focusing on those overexpressing estrogen receptor (ER) or ERBB2/HER2, because these oncogenic receptor pathways are constitutively activated in approximately 80% of all breast cancers. All models demonstrated dose-dependent bortezomib reduction in intracellular 20S activity correlating with cell growth inhibition, and bortezomib IC(50) values (concentrations producing 50% growth inhibition) varied directly with pretreatment 20S activities (r = 0.

Breast cancer growth prevention by statins.

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors.

Validated high-throughput screening of drug-like small molecules for inhibitors of ErbB2 transcription.

A whole cell high-throughput screening assay was developed and tested against > 2,000 structurally and functionally diverse drug-like small molecules to identify lead compounds capable of cell permeability and selective silencing of ErbB2 transcription. Screening employed reporter sublines clonally selected from ErbB2-negative MCF7 breast cancer cells after stable genomic integration of the ErbB2 proximal promoter driving a luciferase reporter; anti-ErbB2 activities (50% inhibitory concentration values) were compared to inhibition of control MCF7 sublines bearing integrated reporters driven by either a mutated ErbB2 promoter or the cyclin D1 promoter. Of the seven resulting lead compounds, four emerged from the National Cancer Institute (NCI)/ Developmental Therapeutics Program (DTP) Structural Diversity Set (NSC-131547, NSC-176328, NSC-259968, and NSC-321237); three others emerged from a panel of anticancer compounds with known mechanistic actions and included a minor groove DNA-binding antibiotic (NSC-58514, chromomycin A3), a hydroxamic acid inhibitor of histone deacetylases (NSC-709238, trichostatin A), and a tripeptide aldehyde proteasome inhibitor (MG-132).

Enhanced pharmacodynamic and antitumor properties of a histone deacetylase inhibitor encapsulated in liposomes or ErbB2-targeted immunoliposomes.

ErbB2-overexpressing human cancers represent potentially sensitive targets for therapy by candidate histone deacetylase (HDAC) inhibitors as we have shown that HDAC inhibitors can selectively reduce ErbB2 expression by repressing the ErbB2 promoter and accelerating the decay of cytoplasmic ErbB2 transcripts. To extend these in vitro findings and enhance the in vivo pharmacodynamic properties of HDAC inhibitors, we stably encapsulated a potent hydroxamate-based HDAC inhibitor (LAQ824) within long-circulating liposomes (Ls-LAQ824) and immunoliposomes (ILs-LAQ824) bearing >10,000 LAQ824 molecules per nanovesicle. Liposomal LAQ824 exhibits prolonged in vivo stability and, unlike free LAQ824, circulates with a half-life of 10.

Activation of nuclear factor-kappaB (NFkappaB) identifies a high-risk subset of hormone-dependent breast cancers.

Activation of nuclear factor-kappaB (NFkappaB) has been linked to the development of hormone-independent, estrogen receptor (ER)-negative human breast cancers. To explore the possibility that activated NFkappaB marks a subset of clinically more aggressive ER-positive breast cancers, NFkappaB DNA-binding was measured in ER-positive breast cancer cell lines and primary breast cancer extracts by electrophoretic mobility shift assay and ELISA-based quantification of specific NFkappaB p50 and p65 DNA-binding subunits. Oxidant (menadione 100 microMx30 min) activation of NFkappaB was prevented by pretreatment with various NFkappaB inhibitors, including the specific IkappaB kinase (IKK) inhibitor, parthenolide (PA), which was found to sensitize MCF-7/HER2 and BT474 but not MCF-7 cells to the antiestrogen tamoxifen.

Thioredoxin and germinating barley: targets and protein redox changes.

The endosperm and embryo of barley ( Hordeum vulgare L.) grain were investigated to relate thioredoxin h and disulfide changes to germination and seedling development. The disulfide proteins of both tissues were found to undergo reduction following imbibition.