Structural Analysis of the Voltage-Dependent Calcium Channel β Subunit Functional Core and Its Complex with the α1 Interaction Domain

AbstractVoltage-dependent calcium channels (VDCC) are multiprotein assemblies that regulate the entry of extracellular calcium into electrically excitable cells and serve as signal transduction centers. The α1 subunit forms the membrane pore while the intracellular β subunit is responsible for trafficking of the channel to the plasma membrane and modulation of its electrophysiological properties. Crystallographic analyses of a β subunit functional core alone and in complex with a α1 interaction domain (AID) peptide, the primary binding site of β to the α1 subunit, reveal that β represents a novel member of the MAGUK protein family. The findings illustrate how the guanylate kinase fold has been fashioned into a protein-protein interaction module by alteration of one of its substrate sites. Combined results indicate that the AID peptide undergoes a helical transition in binding to β. We outline the mechanistic implications for understanding the β subunit's broad regulatory role of the VDCC, particularly via the AID

ERBB receptors in cancer: signaling from the inside

ERBB receptor tyrosine kinases are activated by ligand-induced dimerization followed by activation and transphosphorylation of their intracellular kinase domains. A recent study by Bill and colleagues demonstrates that receptor transphosphorylation can be regulated from inside the cell by members of the cytohesin protein family. These data highlight a novel mechanism of amplification of ERBB receptor signaling output that may contribute to embryogenesis and cancer progression

Cancer targeting with biomolecules: a comparative study of photodynamic therapy efficacy using antibody or lectin conjugated phthalocyanine-PEG gold nanoparticles

The functionalisation of therapeutic nanoparticle constructs with cancer-specific biomolecules can enable selective tumour accumulation and targeted treatment. Water soluble gold nanoparticles (ca. 4 nm) stabilised by a mixed monolayer of a hydrophobic zinc phthalocyanine photosensitiser (C11Pc) and hydrophilic polyethylene glycol (PEG) have been prepared. The C11Pc-PEG gold nanoparticle constructs were further functionalised with jacalin, a lectin specific for the cancer-associated Thomsen–Friedenreich (T) carbohydrate antigen, or with monoclonal antibodies specific for the human epidermal growth factor receptor-2 (HER-2). The two biofunctionalised nanoparticle conjugates produced similar levels of singlet oxygen upon irradiation at 633 nm. Importantly, both nanoparticle conjugates demonstrated extensive, yet comparable, phototoxicity in HT-29 colorectal adenocarcinoma cells (80–90%) and in SK-BR-3 breast adenocarcinoma cells (>99%). Non-conjugated C11Pc-PEG gold nanoparticles were only minimally phototoxic. Lysosomal colocalisation studies performed with the HT-29 colon cancer cells and the SK-BR-3 breast cancer cells revealed that both nanoparticle conjugates were partially localised within acidic organelles, which is typical of receptor-mediated endocytosis. The similarity of the targeted PDT efficacy of the two biofunctionalised C11Pc-PEG gold nanoparticles is discussed with respect to targeting ligand binding affinity and cell surface antigen density as key determinants of targeting efficiency. This study highlights how targeting small cell-surface molecules, such as the T antigen, can mediate a selective photodynamic treatment response which is similar to that achieved when targeting overexpressed protein receptors, such as HER-2. The high prevalence of the T antigen present on the cellular surface of primary tumours emphasises the broad potential applications for lectin-targeted therapies

A Combination of Approved Antibodies Overcomes Resistance of Lung Cancer to Osimertinib by Blocking Bypass Pathways.

<b>Purpose:</b> Because of emergence of resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), no targeted treatments are available for patients with lung cancer who lose sensitivity due to new mutations or bypass mechanisms. We examined in animals and <i>in vitro</i> an alternative therapeutic approach making use of antibodies. <b>Experimental Design:</b> An osimertinib-sensitive animal model of lung cancer, which rapidly develops drug resistance, has been employed. To overcome compensatory hyperactivation of ERK, which we previously reported, an anti-EGFR antibody (cetuximab) was combined with other antibodies, as well as with a subtherapeutic dose of osimertinib, and cancer cell apoptosis was assayed. <b>Results:</b> Our animal studies identified a combination of three clinically approved drugs, cetuximab, trastuzumab (an anti-HER2 mAb), and osimertinib (low dose), as an effective and long-lasting treatment that is able to prevent onset of resistance to osimertinib. A continuous schedule of concurrent treatment was sufficient for effective tumor inhibition and for prevention of relapses. Studies employing cultured cells and analyses of tumor extracts indicated that the combination of two mAbs and a subtherapeutic TKI dose sorted EGFR and HER2 for degradation; cooperatively enhanced apoptosis; inhibited activation of ERK; and reduced abundance of several bypass proteins, namely MET, AXL, and HER3. <b>Conclusions:</b> Our <i>in vitro</i> assays and animal studies identified an effective combination of clinically approved drugs that might overcome resistance to irreversible TKIs in clinical settings. The results we present attribute the long-lasting effect of the drug combination to simultaneous blockade of several well-characterized mechanisms of drug resistance. <i>Clin Cancer Res; 24(22); 5610-21. ©2018 AACR</i> <i>See related commentary by Fan and Yu, p. 5499</i>

MiRNA-513a-5p inhibits progesterone receptor expression and constitutes a risk factor for breast cancer : The hOrmone and Diet in the ETiology of breast cancer prospective study

report was to investigate whether many years before the diagnosis of breast cancer miRNA expression is already disregulated. In order to test this hypothesis, we compared miRNAs extracted from leukocytes in healthy women who later developed breast cancer and in women who remain healthy during the whole 15-year follow-up time. Accordantly, we used a case-control study design nested in the hOrmone and Diet in the ETiology of breast cancer (ORDET) prospective cohort study addressing the possibility that miRNAs can serve as both early biomarkers and components of the hormonal etiological pathways leading to breast cancer development in premenopausal women. We compared leukocyte miRNA profiles of 191 incident premenopausal breast cancer cases and profiles of 191 women who remained healthy over a follow-up period of 20 years. The analysis identified 20 differentially expressed miRNAs in women candidate to develop breast cancer versus control women. The upregulated miRNAs, miR-513-a-5p, miR- 513b-5p and miR-513c-5p were among the most significantly deregulated miRNAs. In multivariate analysis, miR-513a-5p upregulation was directly and statistically significant associated with breast cancer risk (OR = 1.69; 95% CI 1.08-2.64; P = 0.0293). In addition, the upregulation of miR-513-a-5p displayed the strongest direct association with serum progesterone and testosterone levels. The experimental data corroborated the inhibitory function of miR-513a-5p on progesterone receptor expression confirming that progesterone receptor is a target of miR-513a-5p. The identification of upregulated miR-513a-5p with its oncogenic potential further validates the use of miRNAs as long-term biomarker of breast cancer risk

Aberrant transcriptional and post-transcriptional regulation of SPAG5, a YAP-TAZ-TEAD downstream effector, fuels breast cancer cell proliferation

Sperm-associated antigen 5 (SPAG5) is an important driver of the cell mitotic spindle required for chromosome segregation and progression into anaphase. SPAG5 has been identified as an important proliferation marker and chemotherapy-sensitivity predictor, especially in estrogen receptor-negative breast cancer subtypes. Here, we report that SPAG5 is a direct target of miR-10b-3p, and its aberrantly high expression associates with poor disease-free survival in two large cohorts of breast cancer patients. SPAG5 depletion strongly impaired cancer cell cycle progression, proliferation, and migration. Interestingly, high expression of SPAG5 pairs with a YAP/TAZ-activated signature in breast cancer patients. Reassuringly, the depletion of YAP, TAZ, and TEAD strongly reduced SPAG5 expression and diminished its oncogenic effects. YAP, TAZ coactivators, and TEAD transcription factors are key components of the Hippo signaling pathway involved in tumor initiation, progression, and metastasis. Furthermore, we report that SPAG5 is a direct transcriptional target of TEAD/YAP/TAZ, and pharmacological targeting of YAP and TAZ severely reduces SPAG5 expression. Collectively, our data uncover an oncogenic feedback loop, comprising miR-10b-3p, SPAG5, and YAP/TAZ/TEAD, which fuels the aberrant proliferation of breast cancer

HER2 testing in breast cancer: Opportunities and challenges

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-25% of breast cancers, usually as a result of HER2 gene amplification. Positive HER2 status is considered to be an adverse prognostic factor. Recognition of the role of HER2 in breast cancer growth has led to the development of anti-HER2 directed therapy, with the humanized monoclonal antibody trastuzumab (Herceptin (R)) having been approved for the therapy of HER2-positive metastatic breast cancer. Clinical studies have further suggested that HER2 status can provide important information regarding success or failure of certain hormonal therapies or chemotherapies. As a result of these developments, there has been increasing demand to perform HER2 testing on current and archived breast cancer specimens. This article reviews the molecular background of HER2 function, activation and inhibition as well as current opinions concerning its role in chemosensitivity and interaction with estrogen receptor biology. The different tissue-based assays used to detect HER2 amplification and overexpression are discussed with respect to their advantages and disadvantages, when to test (at initial diagnosis or pre-treatment), where to test (locally or centralized) and the need for quality assurance to ensure accurate and valid testing results

Haplotype structure and selection of the MDM2 oncogene in humans

The MDM2 protein is an ubiquitin ligase that plays a critical role in regulating the levels and activity of the p53 protein, which is a central tumor suppressor. A SNP in the human MDM2 gene (SNP309 T/G) occurs at frequencies dependent on demographic history and has been shown to have important differential effects on the activity of the MDM2 and p53 proteins and to associate with altered risk for the development of several cancers. In this report, the haplotype structure of the MDM2 gene is determined by using 14 different SNPs across the gene from three different population samples: Caucasians, African Americans, and the Ashkenazi Jewish ethnic group. The results presented in this report indicate that there is a substantially reduced variability of the deleterious SNP309 G allele haplotype in all three populations studied, whereas multiple common T allele haplotypes were found in all three populations. This observation, coupled with the relatively high frequency of the G allele haplotype in both and Caucasian and Ashkenazi Jewish population data sets, suggests that this haplotype could have undergone a recent positive selection sweep. An entropy-based selection test is presented that explicitly takes into account the correlations between different SNPs, and the analysis of MDM2 reveals a significant departure from the standard assumptions of selective neutrality

Electrochemistry at nanoscale electrodes : individual single-walled carbon nanotubes (SWNTs) and SWNT-templated metal nanowires

Individual nanowires (NWs) and native single-walled carbon nanotubes (SWNTs) can be readily used as well-defined nanoscale electrodes (NSEs) for voltammetric analysis. Here, the simple photolithography-free fabrication of submillimeter long Au, Pt, and Pd NWs, with sub-100 nm heights, by templated electrodeposition onto ultralong flow-aligned SWNTs is demonstrated. Both individual Au NWs and SWNTs are employed as NSEs for electron-transfer (ET) kinetic quantification, using cyclic voltammetry (CV), in conjunction with a microcapillary-based electrochemical method. A small capillary with internal diameter in the range 30–70 μm, filled with solution containing a redox-active mediator (FcTMA+ ((trimethylammonium)methylferrocene), Fe(CN)64–, or hydrazine) is positioned above the NSE, so that the solution meniscus completes an electrochemical cell. A 3D finite-element model, faithfully reproducing the experimental geometry, is used to both analyze the experimental CVs and derive the rate of heterogeneous ET, using Butler–Volmer kinetics. For a 70 nm height Au NW, intrinsic rate constants, k0, up to ca. 1 cm s–1 can be resolved. Using the same experimental configuration the electrochemistry of individual SWNTs can also be accessed. For FcTMA+/2+ electrolysis the simulated ET kinetic parameters yield very fast ET kinetics (k0 > 2 ± 1 cm s–1). Some deviation between the experimental voltammetry and the idealized model is noted, suggesting that double-layer effects may influence ET at the nanoscale