Beta Isoforms Research Articles

Nesprins: tissue-specific expression of epsilon and other short isoforms.

Nesprin-1-giant and nesprin-2-giant regulate nuclear positioning by the interaction of their C-terminal KASH domains with nuclear membrane SUN proteins and their N-terminal calponin-homology domains with cytoskeletal actin. A number of short isoforms lacking the actin-binding domains are produced by internal promotion. We have evaluated the significance of these shorter isoforms using quantitative RT-PCR and western blotting with site-specific monoclonal antibodies. Within a complete map of nesprin isoforms, we describe two novel nesprin-2 epsilon isoforms for the first time. Epsilon isoforms are similar in size and structure to nesprin-1-alpha. Expression of nesprin isoforms was highly tissue-dependent. Nesprin-2-epsilon-1 was found in early embryonic cells, while nesprin-2-epsilon-2 was present in heart and other adult tissues, but not skeletal muscle. Some cell lines lack shorter isoforms and express only one of the two nesprin genes, suggesting that either of the giant nesprins is sufficient for basic cell functions. For the first time, localisation of endogenous nesprin away from the nuclear membrane was shown in cells where removal of the KASH domain by alternative splicing occurs. By distinguishing between degradation products and true isoforms on western blots, it was found that previously-described beta and gamma isoforms are expressed either at only low levels or with a limited tissue distribution. Two of the shortest alpha isoforms, nesprin-1-alpha-2 and nesprin-2-alpha-1, were found almost exclusively in cardiac and skeletal muscle and a highly conserved and alternatively-spliced exon, available in both nesprin genes, was always included in these tissues. These “muscle-specific” isoforms are thought to form a complex with emerin and lamin A/C at the inner nuclear membrane and mutations in all three proteins cause Emery-Dreifuss muscular dystrophy and/or inherited dilated cardiomyopathy, disorders in which only skeletal muscle and/or heart are affected.

Cerebrospinal Fluid Biomarker and Brain Biopsy Findings in Idiopathic Normal Pressure Hydrocephalus

BackgroundThe significance of amyloid precursor protein (APP) and neuroinflammation in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD) is unknown.ObjectiveTo investigate the role of soluble APP (sAPP) and amyloid beta (Aβ) isoforms, proinflammatory cytokines, and biomarkers of neuronal damage in the cerebrospinal fluid (CSF) in relation to brain biopsy Aβ and hyperphosphorylated tau (HPτ) findings.MethodsThe study population comprised 102 patients with possible NPH with cortical brain biopsies, ventricular and lumbar CSF samples, and DNA available. The final clinical diagnoses were: 53 iNPH (91% shunt-responders), 26 AD (10 mixed iNPH+AD), and 23 others. Biopsy samples were immunostained against Aβ and HPτ. CSF levels of AD-related biomarkers (Aβ42, p-tau, total tau), non-AD-related Aβ isoforms (Aβ38, Aβ40), sAPP isoforms (sAPPα, sAPPβ), proinflammatory cytokines (several interleukins (IL), interferon-gamma, monocyte chemoattractant protein-1, tumor necrosis factor-alpha) and biomarkers of neuronal damage (neurofilament light and myelin basic protein) were measured. All patients were genotyped for APOE.ResultsLumbar CSF levels of sAPPα were lower (p<0.05) in patients with shunt-responsive iNPH compared to non-iNPH patients. sAPPβ showed a similar trend (p = 0.06). CSF sAPP isoform levels showed no association to Aβ or HPτ in the brain biopsy. Quantified Aβ load in the brain biopsy showed a negative correlation with CSF levels of Aβ42 in ventricular (r = −0.295, p = 0.003) and lumbar (r = −0.356, p = 0.01) samples, while the levels of Aβ38 and Aβ40 showed no correlation. CSF levels of proinflammatory cytokines and biomarkers of neuronal damage did not associate to the brain biopsy findings, diagnosis, or shunt response. Higher lumbar/ventricular CSF IL-8 ratios (p<0.001) were seen in lumbar samples collected after ventriculostomy compared to the samples collected before the procedure.ConclusionsThe role of sAPP isoforms in iNPH seems to be independent from the amyloid cascade. No neuroinflammatory background was observed in iNPH or AD.

Estrogen receptor beta as a novel target of androgen receptor action in breast cancer cell lines

IntroductionThe two isoforms of estrogen receptor (ER) alpha and beta play opposite roles in regulating proliferation and differentiation of breast cancers, with ER-alpha mediating mitogenic effects and ER-beta acting as a tumor suppressor. Emerging data have reported that androgen receptor (AR) activation inhibits ER-positive breast cancer progression mainly by antagonizing ER-alpha signaling. However, to date no studies have specifically evaluated a potential involvement of ER-beta in the inhibitory effects of androgens.MethodsER-beta expression was examined in human breast cancer cell lines using real-time PCR, Western blotting and small interfering RNA (siRNA) assays. Mutagenesis studies, electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were performed to assess the effects of mibolerone/AR on ER-beta promoter activity and binding.ResultsIn this study, we demonstrate that mibolerone, a synthetic androgen ligand, up-regulates ER-beta mRNA and protein levels in ER-positive breast cancer cells. Transient transfection experiments, using a vector containing the human ER-beta promoter region, show that mibolerone increases basal ER-beta promoter activity. Site-directed mutagenesis and deletion analysis reveal that an androgen response element (ARE), TGTTCT motif located at positions −383 and −377, is critical for mibolerone-induced ER-beta up-regulation in breast cancer cells. This occurs through an increased recruitment of AR to the ARE site within the ER-beta promoter region, along with an enhanced occupancy of RNA polymerase II. Finally, silencing of ER-beta gene expression by RNA interference is able to partially reverse the effects of mibolerone on cell proliferation, p21 and cyclin D1 expression.ConclusionsCollectively, these data provide evidence for a novel mechanism by which activated AR, through an up-regulation of ER-beta gene expression, inhibits breast cancer cell growth.

IL-10 Regulation Of Hyaluronan Synthesis Is Mediated Via TGF-Beta

The fetal scarless phenotype is characterized by an attenuated inflammatory response, an extracellular matrix (ECM) comprised of increased levels of hyaluronan (HA), and elevated levels of TGFB3 compared to TGFB1. We have reported a novel role for the anti-inflammatory cytokine, IL-10, in the fetal regenerative phenotype and its ability to induce scarless healing in adult wounds. Further, we have demonstrated a new non- immunologic mechanism for IL-10, which is regulation of HA. The TGF beta isoforms have also been implicated in HA regulation.

Role of the Coil-Helix Transition within Loop2 in Cardiac Myosin Kinetics Modulation

We have used Molecular Dynamics simulations to examine structural differences of cardiac alpha and beta myosin isoforms, potentially leading to their differences in kinetics. Cardiac myosin isoforms are intensively studied due to correlation between elevated expression level of slower beta isoform and the heart failure. Relationship between kinetic properties of specific isoform and the heart function leads to the new strategy in the failing cardiac muscle treatment, direct myosin activation. Considering myosin head as the potential drug target, it is important to know how different structural elements of the molecule regulate its kinetics.Loop2 (25k-50k loop) connects two domains in myosin heavy chain subfragment S1. Studies of chimeric proteins with interchanged Loop2 showed modulation of protein kinetics and in vitro motility. It was speculated that the major affecter of myosin kinetics is the length of the Loop 2. We have constructed models of alpha and beta cardiac S1 isoforms using available X-ray structure of beta isoform and homology modeling. Trajectories of 0.5us length in explicit solvent for each isoform were calculated. Conformational analysis revealed several regions (including Loop2), adopting significantly different conformations amongst alpha and beta isoforms. The terminal regions of Loop2 in alpha myosin undergo coil-to-helix transitions. This process correlates with the breakdown of salt bridges, coupling Loop2 to the 7-stranded beta sheet in the core of myosin. In contrast, these salt bridges are conserved in the beta myosin isoform. Loop2 is connected to helices attached to P-loop and switch II loop within myosin active site. We conclude that observed coil-to-helix transition within Loop2 of alpha cardiac myosin decouples structural elements of the active site and the 7-stranded beta sheet, thus modulating kinetics of ATP hydrolysis and ADP release. Supported by NIH AR59621.

Protein Kinase C - Its Role in Protrusion Regulation

The PKCs comprise 12 isoforms, and their substrate-specificity is regulated in part by binding to receptors for C kinases (RACKs). Certain isoforms are activated by phorbol 12-myristate 13-acetate (PMA), which also affects shape and adhesion. Different cell types express different subsets of isoforms. In tracheal epithelial cells, the activation of beta and epsilon isoforms by PMA was followed by their degradation. We sought to determine whether such activation-degradation patterns affected protrusion formation or turnover. Cells were treated by transcriptional knockdown (KD) and the prevalence of each protrusion class was analyzed by computerized morphometry. Latent factors for filopodia (#4) and nascent neurites (#7) were calculated based on geometric variables determined for known populations. At zero time, factor #7 was elevated by alpha KD. Control samples, into which a random KD sequence had been introduced, showed an increase in neurites throughout the PMA exposure. During the time course, all KD samples differed from controls at one or more times. Epsilon KD followed by PMA drastically downregulated the isoform by 15 h and decreased #7 values relative to control. Alpha KD was similar suggesting that the early KD effect (increasing #7) was reversed later on. Beta KD cells resembled control at 15 h but alpha KD at the 5-h time. Filopodia were eliminated by a 2-h PMA exposure regardless of the KD agent introduced. Control samples remained depressed, but epsilon and alpha KD samples partially recovered by 5 h. Alpha KD declined again dramatically by 15 h. The time courses suggest that the main effects were exerted by the actin-binding PKC epsilon, but alpha, which is not actin-binding, could have similar effects. The data suggest that competitive binding on RACKs may be occurring, complicating the picture of kinase regulation of adhesion and protrusive activities.

Lysyl Oxidases in the Trabecular Meshwork

The mechanical properties of the extracellular matrix (ECM) play an important role in maintaining cellular function and overall tissue homeostasis. Emerging evidence suggests that biomechanical modifications of the ECM may be initiators and/or drivers of disease, exemplified by increased tissue stiffness. Specific ECM cross-linking enzymes (tissue transglutaminase, lysyl oxidase, and lysyl oxidase-like 1) are expressed in the trabecular meshwork and are regulated by transforming growth factor beta (TGF-β) isoforms. As TGF-β isoforms are elevated in the aqueous humor of glaucoma patients, trabecular meshwork stiffness mediated by ECM cross-linking may be responsible for increased aqueous humor outflow resistance and elevated intraocular pressure.

Integrin αEβ7: molecular features and functional significance in the immune system.

Alpha E beta 7 (αEβ7) is an α-I domain-containing integrin that is highly expressed by a variety of leukocyte populations at mucosal sites including intraepithelial T cells, dendritic cells, mast cells, and T regulatory cells (Treg). Expression depends largely or solely on transforming growth factor beta (TGF-β) isoforms. The best characterized ligand for αEβ7 is E-cadherin on epithelial cells, though there is evidence of a second ligand in the human system. An exposed acidic residue on the distal aspect of E-cadherin domain 1 interacts with the MIDAS site in the αE α-I domain. By binding to E-cadherin, αEβ7 contributes to mucosal specific retention of leukocytes within epithelia. Studies on αE knockout mice have identified an additional important function for this integrin in allograft rejection and have also indicated that it may have a role in immunoregulation. Recent studies point to a multifaceted role for αEβ7 in regulating both innate and acquired immune responses to foreign antigen.

Abstract PD1-3: Ph1b study of the PI3K inhibitor GDC-0032 in combination with fulvestrant in patients with hormone receptor-positive advanced breast cancer

Abstract Background: GDC-0032 is a next-generation PI3K inhibitor with increased anti-tumor activity against PIK3CA mutant cancers. GDC-0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30-fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that GDC-0032 has enhanced activity against PI3K alpha isoform (PIK3CA) mutant breast cancer cell lines. Preclinical data also show enhanced antitumor activity when GDC-0032 is combined with fulvestrant. Material and Methods: A Phase 1b dose escalation study was conducted with evaluation of GDC-0032 doses ranging from 6-9 mg QD in combination with fulvestrant 500mg q4wk (with loading dose of 500mg at day 1, 14 and 28) in a modified 3+3 design. A dose expansion cohort was conducted at the recommended Phase 2 dose of 6 mg QD. Safety and tolerability of GDC-0032 was assessed, as well as pharmacokinetics (PK), pharmacodynamic (PD) assessment of PI3K pathway inhibition by paired tumor biopsies and by FDG-PET, and anti-tumor activity by RECIST. Results: As of 1 Mar 2013, 17 patients were enrolled onto this study with the completion of dose escalation. No dose limiting toxicities (DLTs) were observed at either the 6 mg or 9 mg dose levels. Adverse events (AEs) assessed by the investigator as related to GDC-0032 in ≥10% of patients, were diarrhea, hyperglycemia, stomatitis, fatigue, asthenia, decreased appetite, nausea, mucosal inflammation and rash. No observed apparent PK interactions were observed between GDC-0032 and fulvestrant. The median number of prior systemic therapies was 6. Metabolic partial responses via FDG-PET (≥ 20% decrease in mSUVmax) were observed in 8 out of 11 patients assessed (73%). Confirmed partial responses by RECIST have been observed at both the 6mg and 9mg GDC-0032 dose levels. These include patients who have had prior treatment with fulvestrant. As of 29 May 2013, enrollment onto the dose escalation and expansion cohort has been completed (n = 27). Updated data on safety, pharmacodynamics, efficacy, and biomarker correlates will be presented. Conclusions: The combination of GDC-0032 and fulvestrant is a well-tolerated regimen with promising preliminary efficacy. GDC-0032 is being further investigated in combination with fulvestrant for patients with hormone receptor-positive advanced breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-3.

Proteomic profiling and post-translational modifications in human keratinocytes treated with Mucuna pruriens leaf extract

Ethnopharmacological relevanceMucuna pruriens (Mp) is a plant belonging to the Fabaceae family, with several medicinal properties among which its potential to treat diseases where reactive oxygen species (ROS) play an important role in the pathogeneses. The aim was to investigate the effects of Mp leaf methanolic extract (MPME) on human keratinocytes protein expression and its role in preventing proteins oxidation after oxidative stress (OS) exposure. Material and methodsThe effects of MPME on HaCaT cells protein expression were evaluated treating cells with different concentrations of MPME, with glucose oxidase (GO, source of OS) and with MPME subsequently treated with GO. The protein patterns of treated HaCaT cells are analyzed by two-dimensional gel electrophoresis (2-DE) and compared with that of untreated HaCaT. Immunoblotting was then used to evaluate the role of MPME in preventing the 4-hydroxynonenal protein adducts (4-HNE PAs) formation (marker of OS). ResultsEighteen proteins, identified by mass spectrometry (LC-ESI-CID-MS/MS), were modulated distinctly by MPME in HaCaT. Overall, MPME counteract GO effect, reducing the GO-induced overexpression of several proteins involved in stress response (T-complex protein 1, Protein disulfide-isomerase A3, Protein DJ-1, and Stress-induced-phosphoprotein 1), in cell energy methabolism (Inorganic pyrophosphatase, Triosephosphate isomerase isoform 1, 2-phosphopyruvate-hydratase alpha-enolase, and Fructose-bisphosphate aldolase A isoform 1), in cytoskeletal organization (Cytokeratins 18, 9, 2, Cofilin-1, Annexin A2 and F-actin-capping protein subunit beta isoform 1) and in cell cycle progression (Eukaryotic translation initiation factor 5A-1 isoform B). In addition, MPME decreased the 4-HNE PAs levels, in particular on 2-phosphopyruvate-hydratase alpha-enolase and Cytokeratin 9. ConclusionsOur findings show that MPME might be helpful in the treatment of OS-related skin diseases by preventing protein post-translational modifications (4-HNE PAs).

Toxicoproteomic analysis of bovine aortic endothelial cell under exposure to cigarette smoking extracts

The major health implications of exposure to cigarette smoke that leads to disease development are still being explored. The overall protein expression pattern of bovine aortic endothelial cell (BAEC) has been studied during the treatment with cigarette smoke extract (CSE) by using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Out of an average 950 spots in silver-stained gels, the synthetic levels of 4 proteins were significantly increased, whereas 7 proteins were decreased. The identified proteins have been found to be involved in anti-inflammation (Apolipoprotein A1), energy metabolism (ATP synthase subunit d), signaling pathway (Acetyl-CoA carboxylase; GTP-binding protein), oxidative stress response (Phosphatidylinositol transfer protein beta isoform; phosphatidylinositol transfer protein alpha isoform; Vascular endothelial growth factor), carcinogenesis (Insulin-like growth factor-binding protein 2; Visinin-like protein-1), exocytosis machinery (Synaptosomal-associated protein 25) and immune response (C4b-binding protein). A significantly different protein expression pattern between the control and the CSE-exposed BAEC has been observed and demonstrated on the synthesis level analysis. The systematic approach to the analysis of proteomic responses used as well as the detailed analysis results has been useful in understanding the novel molecular mechanisms for cigarette smoke induced pathological changes. This will help in designing the systemic strategies for the development of new therapeutic approaches to smokerelated diseases.

Changes of calcium/calmodulin-dependent protein kinase II expression in dorsal root ganglia during maturation in long-term diabetes.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered one of the key intracellular signaling proteins for development of neuropathy. We analyzed the expression of total CaMKII (tCaMKII) and its alpha, beta, gamma and delta isoforms in dorsal root ganglia (DRG) in a rat model of Diabetes mellitus type I (DM1), 6 months and 1 year after diabetes induction. Diabetes was induced with streptozotocin and confirmed by measuring glucose levels and weight increase. Immunohistochemistry was performed for detection of tCaMKII and its isoforms in L4 and L5 DRGs. A significant decrease of CaMKII alpha and beta isoforms was noted 6 months after diabetes induction, while CaMKII gamma and delta were significantly decreased after 12 months in diabetic rats compared to controls. Analysis of neuronal subgroups based on the neuronal diameter revealed that the expression of alpha, beta and delta isoforms decreased only in small-diameter neurons. In conclusion, a significant decrease of specific CaMKII isoforms in small-diameter DRG neurons may suggest involvement of CaMKII alpha, beta and delta in the development of complex events responsible for the development of neuropathy in long-term diabetes during maturation. CaMKII is a part of the neuronal pathway that regulates the firing properties of excitable cells, especially neurons, and decreased CaMKII activity may be responsible for generation of aberrant signals, hyperalgesia and neuropathic pain.

PI3K-Alpha Plays A Major Role In Multiple Myeloma and Its Inhibition With BYL917 Decreases Proliferation, Synergizes With Other Therapies and Overcomes Stroma-Induced Resistance

IntroductionMultiple myeloma (MM) is the second most prevalent hematologic malignancy and is still incurable. The PI3K pathway is activated and correlated with drug resistance in MM. Pan-inhibition of the PI3K pathway resulted in serious side effects, therefore, we focused on specific inhibition of the PI3K-alpha isoform. We investigated the role of PI3K- alpha in the progression and drug resistance of MM, by inhibiting the PI3K- alpha with a novel specific inhibitor BYL719. This is the first study to describe the preclinical effect of BYL719 on MM in particular and in hematologic malignancies in general. Methods and ResultsWe analyzed the gene expression of PI3K isoforms in MM patients based on published datasets from the Gene Expression Omnibus by Zhan et al and found that PI3K-alpha and beta isoforms were highly expressed in MM. However, the fold-change of expression of the alpha isoform in MM patients was higher than the beta isoform, compared to healthy subjects. BYL719 inhibited the survival of MM cells isolated from three MM patients by MTT, at an IC50 around 1uM, but had no effect on normal PBMCs. Furthermore, BYL719 inhibited survival of all MM cell lines in a different manner. In silico-predicted activity of PI3K-alpha in the cell lines was exponentially correlated with the killing induced by the PI3K inhibitor BYL719 in vitro. BYL719 significantly decreased the activation of the PI3K signaling related proteins (pAKT, pS6R, and pGSK) by western blotting. Moreover, BYL719 inhibited cell cycle of MM cells detected by PI staining showed induction of G1-phase cell cycle arrest. BYL719 inhibited MM proliferation of MM1s cells in a dose dependent manner, through decreasing the levels of pCyclin-E1 and pRb, and increasing of P27 levels. The results were in agreement with the in silico studies that predicted the inhibition of other cell-cycle proteins including CDK4-Cyclin D complex, Myc-Max complex and CDK1-Cyclin B complex by BYL719 in a dose dependent manner. Moreover, BYL719 (0-2.5uM) increased the fraction of apoptotic MM cells in a dose depended manner, as detected by Annexin/PI staining. BYL719 induced apoptosis signaling by inducing the cleavage of Caspase-3, Caspase-9 and PARP by western blotting, in a dose dependent manner. We tested the effect of the combination of BYL719 with other drugs (Bortezomib and Carfilzomib) on survival of MM cells by MTT. We found that the combination of the two drugs decreased the surviving fraction of MM cells more than each of the drugs alone. Mechanistically, Bortezomib increased pAKT and pS6R as a resistance mechanism, and BYL719 abolished the Bortezomib-induced increase of pAKT and pS6R. Moreover, BYL719 enhanced the activation of pJNK induced by Carfilzomib and the combination of each drug increased cleavage of PARP, caspase-3 and caspase-9 more than each of the drugs alone. We tested the effect of BYL719 on the interaction of MM cells with BM stromal cells (BMSCs), and it was found that BYL719 decreased the adhesion of MM cells to BMSCs in a dose-dependent manner. Mechanistically, BYL719 decreased the activation of adhesion signaling such as pFAK, pSRC and pCofilin in a dose dependent manner. The in silico studies predicted the inhibition of the small GTPases Rho, Rac and Cdc42. To test the effect of BYL719 on drug resistance induced by the BM stroma, MM cells were co-Cultured with BMSCs, treated with BYL719 in combination with Bortezomib or Carfilzomib. It was found that co-culture with BMSCs increased the surviving fraction of MM cells after treatment with Bortezomib and Carfilzomib, as a drug resistance mechanism. The combination of the two drugs with BYL719 overcame the resistance induced by the stroma and reduced the surviving fraction to the values observed for treatment without presence of stroma. ConclusionThis is the first study to describe preclinical effect of BYL719 on MM in particular and hematologic malignancies in general. The PI3K-alpha isoform plays a major role in the progression and drug resistance in MM cells, and it's inhibition with BYL719 reduces proliferation, inhibits cell cycle and induces apoptosis in MM cells. Moreover, it showed that BYL719 synergizes with Bortezomib and Carfilzomib, and overcomes drug resistance induced by BM stroma. Our findings provide a preclinical basis of future clinical trial of BYL719 in MM as a single agent or in combination with other drugs. Disclosures:Vali:Cellworks Group Inc., San Jose, CA, USA: Employment. Abbasi:Cellworks Group Inc: Employment.

Neuregulin as a Heart Failure Therapy and Mediator of Reverse Remodeling

The beta isoform of Neuregulin-1 (NRG-1β), along with its receptors (ErbB2-4), is required for cardiac development. NRG-1β, as well as the ErbB2 and ErbB4 receptors, is also essential for maintenance of adult heart function. These observations have led to its evaluation as a therapeutic for heart failure. Animal studies and ongoing clinical trials have demonstrated beneficial effects of two forms of recombinant NRG-1β on cardiac function. In addition to the possible role for recombinant NRG-1βs as heart failure therapies, endogenous NRG-1β/ErbB signaling appears to play a role in restoring cardiac function after injury. The potential mechanisms by which NRG-1β may act as both a therapy and a mediator of reverse remodeling remain incompletely understood. In addition to direct effects on cardiac myocytes NRG-1β acts on the vasculature, interstitium, cardiac fibroblasts, and hematopoietic and immune cells, which, collectively, may contribute to NRG-1β's role in maintaining cardiac structure and function, as well as mediating reverse remodeling.

Abstract B218: Drug-drug interaction assessment of GDC-0032, a beta-sparing inhibitor of phosphoinositide 3-kinase (PI3K), using midazolam, a sensitive CYP3A4 substrate.

Abstract Background: GDC-0032 is an orally-bioavailable, potent, and selective inhibitor of Class IA PI3K alpha, delta, gamma, and p110 beta isoforms, with 30 fold less inhibition of the p110 beta isoform. GDC-0032 has shown promising preliminary clinical activity in PIK3CA-mutant cancers and is being explored in solid tumors and in combination with endocrine therapies in breast cancer. In vitro, GDC 0032 was not a potent inhibitor of CYP isoforms 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4 (IC50 &amp;gt; 30 µM) but displayed weak time-dependent inhibition of CYP3A4, with KI and kinact values of 77 µM and 0.030 min-1, respectively. In addition, GDC-0032 induced CYP3A4, but not CYP1A2 or CYP2B6 in human hepatocytes. Based on these data, the potential for GDC-0032 to impact the pharmacokinetics (PK) of midazolam (MDZ), a sensitive in vivo marker of CYP3A4 enzymatic activity, was assessed in a Phase I trial in patients with cancer. Methods: Twelve patients with advanced or metastatic solid tumors were enrolled. All patients received 5 mg MDZ on Day 1 (D1), followed by continuous daily dosing of GDC-0032 (9 mg) starting on D2. A second MDZ dose was given on D16. Predose, 0.5, 1, 1.5, 2, 3, 4, 8, and 24 hr post-dose plasma samples were collected on D1 and D16. Maximum concentration (Cmax) and area under the concentration-time curve (AUC0-24) of MDZ were estimated by non-compartmental methods using WinNonlin (version 5.2.1). MDZ plasma Cmax and AUC0-24 without GDC-0032 (D1) and after 15 daily doses of GDC-0032 (D16) were compared. The geometric mean ratios (GMRs) of Cmax and AUC0-24 and the corresponding 90% CIs were estimated (WinNonlin version 5.2.1). Physiologically-Based Pharmacokinetic (PBPK) modeling was used to prospectively predict the DDI potential of GDC-0032 (Simcyp version 11.0 and Gastroplus version 8.0). Results: No trend in individual Cmax or AUC0-24 of MDZ was observed post-GDC-0032 treatment relative to pre-treatment. The change in MDZ exposure (Cmax and AUC0-24) before and after GDC-0032 treatment was less than 2-fold for all patients (median: 1.1 fold; range: 0.4-1.9). The GMRs (90% CIs) for MDZ+GDC-0032 relative to MDZ alone for Cmax and AUC0-24 were 0.98 (0.70-1.37) and 1.04 (0.68-1.60), respectively. Prospective PBPK modeling accurately predicted a lack of marked DDI between GDC-0032 and MDZ (1-1.4 fold change). Conclusions: Despite in vitro results suggesting that GDC-0032 may impact the PK of concomitant medications which are metabolized by CYP3A4, co-administration with MDZ did not change the PK of MDZ in patients with cancer. PBPK modeling predicted little to no impact of GDC-0032 on the PK of MDZ, consistent with the in vivo observation. These results indicate that GDC-0032 may be given concomitantly with CYP substrates with minimal risk of a PK drug-drug interaction. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B218. Citation Format: Richard A. Graham, Sravanthi Cheeti, Laurent Salphati, Tong Lu, Carla Kurkjian, Daniel Von Hoff, Jasgit Sachdev, Dejan Juric, Jose Baselga, Hema Parmer, Jerry Hsu, Jeffrey Infante. Drug-drug interaction assessment of GDC-0032, a beta-sparing inhibitor of phosphoinositide 3-kinase (PI3K), using midazolam, a sensitive CYP3A4 substrate. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B218.

Estrogen Receptor Isoform Beta Is Expressed in Rectosigmoid Endometriosis and Its Adjacent Muscular Tissue Independently of the Phase of the Menstrual Cycle

Estrogen Receptor Isoform Beta Is Expressed in Rectosigmoid Endometriosis and Its Adjacent Muscular Tissue Independently of the Phase of the Menstrual Cycle

Abstract 626: Caveolin-1 (CAV1) Levels Determine the Cell Surface Expression of the Dopamine D1-like Receptors

In human renal proximal tubule cells, the dopamine-1 receptor (D1R) increases cAMP production and negatively regulates sodium transport. Defects in D1R signaling have been associated with SNPs in G-protein coupled receptor kinase type 4 (GRK4), which hyperphosphorylates and decreases D1R function, leading to sodium retention. We have previously shown that knockdown of CAV1 in proximal tubule cells impairs sodium transport. In this report we show that CAV1 reduction, knockdown or knockout leads to decreased dopamine D1-like receptor binding at the proximal tubule cell surface. Dopamine D1-like brush border and cell surface binding was measured using Bodipy-FL conjugated SKF83566 . WT and Caveolin 1 knockout (CAV1KO) kidney cortex brush border membrane isolation was performed by magnetic lotus tetragonolobus agglutinin affinity purification and fluorescent SKF83566 binding by microplate fluorometry. CAV1KO had a 35.8±3.3% decrease in binding vs WT (p&lt;0.01, n=6 per group, WT level 18,492 RFU above background). In hTERT immortalized human renal proximal tubule cell lines, lentiviral shRNAi CAV1 stable knockdown cell lines had a 76.4±2.5%, p&lt;0.001 n=4, reduction in CAV1 expression by western blot. Cell surface D1-like receptor binding was reduced by 34.0±1.3% (p&lt;0.001, n=4) in the CAV1 knockdown cell lines (scrambled control levels 117193 RFU). We previously have shown that CAV1 expression is dramatically reduced in the hypertensive SHR proximal tubule cell line vs the normotensive WKY proximal tubule cell line. The SHR cell line had a 57.5±1.2% (p&lt;0.001, n=7) reduction in D1-like receptor binding (WKY 116114 RFU). Re-expression of rat CAV1 alpha or CAV1 beta isoform in the SHR cell line by stable lentiviral infection increased binding by 51.6±2.1%, and 63.3±1.6% respectively (p&lt;0.01, n=14). Sodium transport was decreased by CAV1 alpha and CAV1 beta expression in SHR cell lines. The average number of domes per 10x objective fields of view was 9.5±1.6 in SHR cell, 4.5±0.3 in SHR CAV1alpha (p&lt;0.05, n=4 vs SHR) and in SHR CAV1beta 5.0±0.3 (p&lt;0.05, n=4 vs SHR). In mouse, rat, and human renal proximal tubule cells, CAV1 is necessary for dopamine D1-like receptor cell surface localization and for normal dopaminergic inhibition of sodium transport.

Multiple host kinases contribute to Akt activation during Salmonella infection.

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3–5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3–5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

Comparative proteomic analysis of neutrophils from patients with microscopic polyangiitis and granulomatosis with polyangiitis

Both microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) belong to ANCA-associated vasculitis (AAV), in which neutrophils play a key role in their pathology. In this study, in order to discriminate between MPA and GPA, protein profiles of peripheral blood polymorphonuclear cells (PMNs) of 11 MPA patients and 9 GPA patients and 10 healthy controls (HC) were analyzed by 2D-DIGE. In all the 864 spots detected, intensity of 55 spots was significantly different (p<0.05) among the three groups by ANOVA. 31 out of the 55 spots were identified by mass spectrometry. Orthogonal partial-least-squares-discriminate analysis revealed that the abundance profile of the protein spots discriminated the AAV group from the HC group, and the MPA group from the GPA group completely. 13 protein spots were considered as biomarker candidates to distinguish between MPA and GPA. In those, spots whose intensity was higher in MPA than in GPA included actin with various pI values, while a considerable part of spots whose intensity was higher in GPA were proteins related with the activity of neutrophils. Among the candidate proteins, ROC analysis showed that a combination of neutrophil gelatinase-associated lipocalin and a-kinase anchor protein 7 isoforms beta had a high diagnostic potential. In this study, protein profiles of polymorphonuclear cells (PMNs) of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients and healthy controls (HC) were investigated by 2D-DIGE, and MS analysis. As a result, we found that the protein profiles of PMNs were useful for distinguishing between patients (MPA and GPA) and HC, and between patients with MPA and patients with GPA. Especially, we found that the 13 protein spots that consisted of 10 proteins considerably contributed to the discrimination between MPA and GPA. This is the first to demonstrate that protein profiles of PMNs are different among MPA, GPA and healthy control. The 10 proteins we identified in this study would be new biomarkers for the diagnosis of the diseases, and may be reflect the pathology difference between MPA and GPA.

Differential expression of estrogen receptor beta isoforms in prostate cancer through interplay between transcriptional and translational regulation

Estrogen receptor β (ERβ) and its isoforms have different putative functions and expression patterns in prostate cancer. Current studies on 5′-most exons, 0K and 0N, show that their respective promoters are actively involved in transcription. These data, however, do not explain why ERβ isoforms are differentially expressed in normal and cancerous tissues, since 0K and 0N transcripts are detectable in clinical specimens. Various combinations of 5′ untranslated exons, termed exon 0Xs, associate with promoter 0K only and exon 0Xs accommodate upstream open reading frames (uORFs) reducing protein expression. Moreover, ERβ1, 2, and 5 are transcriptionally linked to promoter 0K; exon 0Xs are spliced only into ERβ2 and ERβ5 transcripts, suggesting that their expressions are regulated post-transcriptionally by exon 0Xs. This study reveals that expression of ERβ1 is regulated primarily at the transcriptional level, whereas that of ERβ2 and ERβ5 is controlled by the interplay between transcriptional and post-transcriptional regulation.