Adva Yeheskel

D538G Mutation in Estrogen Receptor-α: A Novel Mechanism for Acquired Endocrine Resistance in Breast Cancer

Resistance to endocrine therapy occurs in virtually all patients with estrogen receptor α (ERα)-positive metastatic breast cancer, and is attributed to various mechanisms including loss of ERα expression, altered activity of coregulators, and cross-talk between the ERα and growth factor signaling pathways. To our knowledge, acquired mutations of the ERα have not been described as mediating endocrine resistance. Samples of 13 patients with metastatic breast cancer were analyzed for mutations in cancer-related genes. In five patients who developed resistance to hormonal therapy, a mutation of A to G at position 1,613 of ERα, resulting in a substitution of aspartic acid at position 538 to glycine (D538G), was identified in liver metastases. Importantly, the mutation was not detected in the primary tumors obtained prior to endocrine treatment. Structural modeling indicated that D538G substitution leads to a conformational change in the ligand-binding domain, which mimics the conformation of activated ligand-bound receptor and alters binding of tamoxifen. Indeed, experiments in breast cancer cells indicated constitutive, ligand-independent transcriptional activity of the D538G receptor, and overexpression of it enhanced proliferation and conferred resistance to tamoxifen. These data indicate a novel mechanism of acquired endocrine resistance in breast cancer. Further studies are needed to assess the frequency of D538G-ERα among patients with breast cancer and explore ways to inhibit its activity and restore endocrine sensitivity.

Multiple congenital anomalies-hypotonia-seizures syndrome is caused by a mutation in PIGN

Background This study reports on a hitherto undescribed autosomal recessive syndrome characterised by dysmorphic features and multiple congenital anomalies together with severe neurological impairment, chorea and seizures leading to early death, and the identification of a gene involved in the pathogenesis of the disease. Methods Homozygosity mapping was performed using Affymetrix Human Mapping 250k NspI arrays. Sequencing of all coding exons of the candidate genes was performed with primer sets designed using the Primer3 program. Fluorescence activated cell sorting was performed using conjugated antibody to CD59. Staining, acquisition and analysis were performed on a FACSCalibur flow cytometer. Results Using homozygosity mapping, the study mapped the disease locus to 18q21.32–18q22.1 and identified the disease-causing mutation, c.2126G→A (p.Arg709Gln), in PIGN, which encodes glycosylphosphatidylinositol (GPI) ethanolamine phosphate transferase 1, a protein involved in GPI-anchor biosynthesis. Arginine at the position 709 is a highly evolutionarily conserved residue located in the PigN domain. The expression of GPI linked protein CD59 on fibroblasts from patients as compared to that in a control individual showed a 10-fold reduction in expression, confirming the pathogenic consequences of the mutation on GPI dependent protein expression. Conclusions The abundant expression of PIGN in various tissues is compatible with the diverse phenotypic features observed in the patients and with the involvement of multiple body systems. The presence of developmental delay, hypotonia, and epilepsy combined with multiple congenital anomalies, especially anorectal anomalies, should lead a clinician to suspect a GPI deficiency related disorder.

Insulin-like Growth Factor-I Receptor (IGF-IR) Translocates to Nucleus and Autoregulates IGF-IR Gene Expression in Breast Cancer Cells

The insulin-like growth factor (IGF) system plays an important role in mammary gland biology as well as in the etiology of breast cancer. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I and IGF-II, has emerged in recent years as a promising therapeutic target. The IGF and estrogen signaling pathways act in a synergistic manner in breast epithelial cells. The present study was aimed at investigating 1) the putative translocation of IGF-IR and the related insulin receptor (IR) to the nucleus in breast cancer cells, 2) the impact of IGF-IR and IR levels on IGF-IR biosynthesis in estrogen receptor (ER)-positive and ER-depleted breast cancer cells, and 3) the potential transcription factor role of IGF-IR in the specific context of IGF-IR gene regulation. We describe here a novel mechanism of autoregulation of IGF-IR gene expression by cellular IGF-IR, which is seemingly dependent on ER status. Regulation of the IGF-IR gene by IGF-IR protein is mediated at the level of transcription, as demonstrated by 1) binding assays (DNA affinity chromatography and ChIP) showing specific IGF-IR binding to IGF-IR promoter DNA and 2) transient transfection assays showing transactivation of the IGF-IR promoter by exogenous IGF-IR. The IR is also capable of translocating to the nucleus and binding the IGF-IR promoter in ER-depleted, but not in ER-positive, cells. However, transcription factors IGF-IR and IR display diametrically opposite activities in the context of IGF-IR gene regulation. Thus, whereas IGF-IR stimulated IGF-IR gene expression, IR inhibited IGF-IR promoter activity. In summary, we have identified a novel mechanism of IGF-IR gene autoregulation in breast cancer cells. The clinical implications of these findings and, in particular, the impact of IGF-IR/IR nuclear localization on targeted therapy require further investigation.

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

Summary Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60–269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

DOC2B, C2 Domains, and Calcium: A Tale of Intricate Interactions

Ca+2-dependent exocytosis involves vesicle docking, priming, fusion, and recycling. This process is performed and regulated by a vast number of synaptic proteins and depends on proper protein–protein and protein–lipid interactions. Double C2 domain (DOC2) is a protein family of three isoforms found while screening DNA libraries with a C2 probe. DOC2 has three domains: the Munc13-interacting domain and tandem C2s (designated C2A and C2B) connected by a short polar linker. The C2 domain binds phospholipids in a Ca2+-dependent manner. This review focuses on the ubiquitously expressed isoform DOC2B. Sequence alignment of the tandem C2 protein family in mouse revealed high homology (81%) between rabphilin-3A and DOC2B proteins. We created a structural model of DOC2Bs C2A based on the crystal structure of rabphilin-3A with and without calcium and found that the calcium-binding loops of DOC2B move upon calcium binding, enabling efficient plasma membrane penetration of its C2A. Here, we discuss the potential relation between the DOC2B bioinformatical model and its function and suggest a possible working model for its interaction with other proteins of the exocytotic machinery, including Munc13, Munc18, and syntaxin.

The gene road to royalty – differential expression of hydroxylating genes in the mandibular glands of the honeybee

The advances in honeybee sociogenomics have paved the way for the study of social communication processes at the gene level, in particular the expression of caste‐specific pheromones. The queen honeybee mandibular pheromone provides an excellent model system, in that biosynthesis of the hydroxylating fatty acid caste‐specific pheromone appears to be reduced to a single chemical hydroxylation step of stearic acid. Queens are typified by ω‐1‐hydroxylation, as opposed to the worker‐typical ω‐hydroxylation. We hypothesized that this bifurcation is the consequence of differential expression of caste‐specific genes that code for fatty acid‐hydroxylating enzymes from the cytochrome P450 (CYP) family. Bioinformatics studies disclosed two candidate proteins CYP4AA1 and CYP18A1. We thus investigated the expression of these genes in the mandibular glands of queens, and of queenright (QR) and queenless (QL) workers. The real‐time PCR results revealed that CYP4AA1 (ω‐hydroxylation) was expressed at high levels in both QR and QL workers, whereas in queens its expression was negligible. The expression of CYP18A1 (ω‐1‐hydroxylation), on the other hand, was high in the queen’s glands and negligible in those of QR workers. In QL workers, however, the expression of CYP18A1 was considerably elevated and significantly greater than in QR workers. Three‐dimensional structural models constructed for these enzymes demonstrate differences in the active site between CYP18A1 and CYP4AA1, in line with their differential catalytic specificity. The fact that queen pheromone plasticity can be tracked all the way to gene expression provides a new insight into the process of caste differentiation and the accompanying social communication.

Sexual divergence in microtubule function: the novel intranasal microtubule targeting SKIP normalizes axonal transport and enhances memory.

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, is a frequent autism spectrum disorder (ASD)-mutated gene. ADNP associates with microtubule end-binding proteins (EBs) through its SxIP motif, to regulate dendritic spine formation and brain plasticity. Here, we reveal SKIP, a novel four-amino-acid peptide representing an EB-binding site, as a replacement therapy in an outbred Adnp-deficient mouse model. We discovered, for the first time, axonal transport deficits in Adnp+/− mice (measured by manganese-enhanced magnetic resonance imaging), with significant male–female differences. RNA sequencing evaluations showed major age, sex and genotype differences. Function enrichment and focus on major gene expression changes further implicated channel/transporter function and the cytoskeleton. In particular, a significant maturation change (1 month-five months) was observed in beta1 tubulin (Tubb1) mRNA, only in Adnp+/+ males, and sex-dependent increase in calcium channel mRNA (Cacna1e) in Adnp+/+ males compared with females. At the protein level, the Adnp+/− mice exhibited impaired hippocampal expression of the calcium channel (voltage-dependent calcium channel, Cacnb1) as well as other key ASD-linked genes including the serotonin transporter (Slc6a4), and the autophagy regulator, BECN1 (Beclin1), in a sex-dependent manner. Intranasal SKIP treatment normalized social memory in 8- to 9-month-old Adnp+/−-treated mice to placebo-control levels, while protecting axonal transport and ameliorating changes in ASD-like gene expression. The control, all d-amino analog D-SKIP, did not mimic SKIP activity. SKIP presents a novel prototype for potential ASD drug development, a prevalent unmet medical need.

Tumor Suppressor Activity of Klotho in Breast Cancer is Revealed by Structure-function Analysis

Klotho is a transmembrane protein containing two internal repeats, KL1 and KL2, both displaying significant homology to members of the β-glycosidase family. Klotho is expressed in the kidney, brain, and various endocrine tissues, but can also be cleaved and act as a circulating hormone. Klotho is an essential cofactor for binding of fibroblast growth factor 23 (FGF23) to the FGF receptor and can also inhibit the insulin-like growth factor-1 (IGF-1) pathway. Data from a wide array of malignancies indicate klotho as a tumor suppressor; however, the structure–function relationships governing its tumor suppressor activities have not been deciphered. Here, the tumor suppressor activities of the KL1 and KL2 domains were examined. Overexpression of either klotho or KL1, but not of KL2, inhibited colony formation by MCF-7 and MDA-MB-231 cells. Moreover, in vivo administration of KL1 was not only well tolerated but significantly slowed tumor formation in nude mice. Further studies indicated that KL1, but not KL2, interacted with the IGF-1R and inhibited the IGF-1 pathway. Based on computerized structural modeling, klotho constructs were generated in which critical amino acids have been mutated. Interestingly, the mutated proteins retained their tumor suppressor activity but showed reduced ability to modulate FGF23 signaling. These data indicate differential activity of the klotho domains, KL1 and KL2, in breast cancer and reveal that the tumor suppressor activities of klotho can be dissected from its physiologic activities. Implications: These findings pave the way for a rational design of safe klotho-based molecules for the treatment of breast cancer. Mol Cancer Res; 13(10); 1398–407. ©2015 AACR.

Genetic and antigenic characterization of sigma C protein from avian reovirus.

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine.

Activity-Dependent Neuroprotective Protein (ADNP): A Case Study for Highly Conserved Chordata-Specific Genes Shaping the Brain and Mutated in Cancer

The recent finding of activity-dependent neuroprotective protein (ADNP) as a protein decreased in serum of patients with Alzheimers disease (AD) compared to controls, alongside with the discovery of ADNP mutations in autism and coupled with the original description of cancer mutations, ignited an interest for a comparative analysis of ADNP with other AD/autism/cancer-associated genes. We strive toward a better understanding of the molecular structure of key players in psychiatric/neurodegenerative diseases including autism, schizophrenia, and AD. This article includes data mining and bioinformatics analysis on the ADNP gene and protein, in addition to other related genes, with emphasis on recent literature. ADNP is discovered here as unique to chordata with specific autism mutations different from cancer-associated mutation. Furthermore, ADNP exhibits similarities to other cancer/autism-associated genes. We suggest that key genes, which shape and maintain our brain and are prone to mutations, are by in large unique to chordata. Furthermore, these brain-controlling genes, like ADNP, are linked to cell growth and differentiation, and under different stress conditions may mutate or exhibit expression changes leading to cancer propagation. Better understanding of these genes could lead to better therapeutics.