Beta Isoforms Research Articles

Abstract 3741: Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma

Abstract Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome. Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.

Abstract 1871: Alpha and beta isoforms of fibroblast growth factor receptor 1 in prostate cancer

Abstract Castrate-resistant progression and bone metastases are hallmarks of advanced prostate cancer (PCa). The fibroblast growth factor (FGF)/FGF receptor (FGFR) complex mediates tumor-stromal interactions and is commonly altered in PCa. FGFR1, FGFR2, FGFR3, and FGFR4 genes encode alternatively spliced variants of FGFRs that vary in the extracellular ligand-binding and intracellular kinase domains. A published study from our group implicated FGFR1 as a therapy target for PCa bone metastases (STM 2014; 6:252ra122). Further, our studies of FGFR1 transcripts by RNA sequencing of 183 human PCas identified eight different protein coding transcripts as the most abundantly expressed with different human PCas expressing different FGFR1 isoforms (Abstract #3913 AACR 2015). These results suggest that different FGFR1 isoforms in PCa cells may partially underlie the biological heterogeneity of PCa. The studies presented here focus in two of the best-characterized isoforms: FGFR1alpha (R1alpha), with 3 Ig-like domains, and 822 aa in length; and FGFR1beta (R1beta), with only 2 Ig-like domains and 733 aa in length. We assessed whether these isoforms induce activation of the same pathways using PC3 cells transiently transfected with empty vector (EV), R1alpha (NM_023110.2) or R1beta (NM_023105.2) and treated with vehicle, FGF2 or FGF9. By Western blot analysis we found that total FGFR1 expression (relative to a loading control) was similar in cells transfected with R1beta or R1alpha. Levels of p-FGFR1 were high in untreated cells transfected with R1alpha, but no further induction was observed after treatment with FGF2 or FGF9. However, p-FGFR1 expression was almost undetectable in untreated cells expressing R1beta and was slightly induced by FGF2 but not by FGF9. p-PLCγ expression was found only in cells expressing R1alpha. We subsequently stably transfected these isoforms in PC3 cells and discovered that while no significant difference in R1alpha and R1beta transcript levels was detected, the levels of R1alpha protein were higher than those of R1beta suggesting that these isoforms may undergo different translational regulation, We also performed in vivo studies by subcutaneous injection of R1alpha or R1beta expressing PC3 cells in immunocompromised mice and weekly monitored tumor volume. We found that PC3 cells expressing R1alpha developed significantly larger tumors than PC3 cells expressing R1beta. We are now in the process of analyzing at the molecular level these tissue specimens. In conclusion, R1alpha and R1beta isoforms trigger different biological effects in PCa cells. Because different isoforms are expressed in different prostate tumors, the expression of these isoforms could be associated with the typical PCa heterogeneity and could explain differences in therapy responses to FGFR1 blockade. These results warrant further studies to fully understand the biological implications of FGFR1 isoforms in the pathogenesis of PCa. Citation Format: Estefania Labanca, Xinhai Wan, Jun Yang, Matthew Iyer, Christopher Logothetis, Arul Chinnaiyan, Nora Navone. Alpha and beta isoforms of fibroblast growth factor receptor 1 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1871.

Abstract 372: Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (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. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013). To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to taselisib, we profiled over 550 cell lines, encompassing 13 of the main tissue types, to increasing concentrations of taselisib. As expected, a small percentage of all tumor types responded to taselisib and correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 0.5uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Interestingly, the majority of HNSCC cell lines displayed an activated PI3K pathway as evidenced by phosphorylation of key proteins, such as AKT, S6 and PRAS40 implicating that downstream phospho-protein analysis of the PI3K pathway may not predict for sensitivity to taselisib. Mutational profiling identified that only 3 out of the tested 31 HNSCC cell lines harbored a mutation within PIK3CA, suggesting that additional genotypes may explain the sensitivity to taselisib. Additional molecular analysis assessing both gene expression and copy number levels of genes relevant to the PI3K pathway found that many of the taselisib sensitive cell lines had an activated ERBB signaling node through low level amplification of ERBB receptors (e.g. EGFR, FGFR1) or via a NRG1:ERBB3 autocrine mechanism. As many ERBB receptors heterodimerize with ERBB3, which contains p85 binding sites and potentially activates the PI3K pathway, we next tested whether taselisib would enhance the potency of ERBB receptor inhibitors in biomarker-defined subset of HNSCC cell lines. Combination screens were performed in cell lines with amplified EGFR, ERBB2, and NRG1:ERBB3 autocrine signaling with taselisib + tarceva or lapatinib. We found that combination effects assessed using the BLISS excess method showed a synergistic interaction. These results suggest that taselisib may have therapeutic potential for the treatment of HNSCC. Citation Format: Heidi M. Savage, Carol O’Brien, Heather Moore, Mark R. Lackner, Robert Yauch, Jeffrey Settleman, Timothy R. Wilson. Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 372.

Signalling by Transforming Growth Factor Beta Isoforms in Wound Healing and Tissue Regeneration

Transforming growth factor beta (TGFβ) signalling is essential for wound healing, including both non-specific scar formation and tissue-specific regeneration. Specific TGFβ isoforms and downstream mediators of canonical and non-canonical signalling play different roles in each of these processes. Here we review the role of TGFβ signalling during tissue repair, with a particular focus on the prototypic isoforms TGFβ1, TGFβ2, and TGFβ3. We begin by introducing TGFβ signalling and then discuss the role of these growth factors and their key downstream signalling mediators in determining the balance between scar formation and tissue regeneration. Next we discuss examples of the pleiotropic roles of TGFβ ligands during cutaneous wound healing and blastema-mediated regeneration, and how inhibition of the canonical signalling pathway (using small molecule inhibitors) blocks regeneration. Finally, we review various TGFβ-targeting therapeutic strategies that hold promise for enhancing tissue repair.

The molecular, temporal and region-specific requirements of the beta isoform of Calcium/Calmodulin-dependent protein kinase type 2 (CAMK2B) in mouse locomotion.

Genetic approaches using temporal and brain region-specific restricted gene deletions have provided a wealth of insight in the brain regions and temporal aspects underlying spatial and associative learning. However, for locomotion such extensive studies are still scarce. Previous studies demonstrated that Camk2b–/– mice, which lack the β isoform of Calcium/Calmodulin-dependent protein kinase 2 (CAMK2B), show very severe locomotion deficits. However, where these locomotion deficits originate is unknown. Here we made use of novel Camk2b mutants (Camk2bf/f and Camk2bT287A), to explore the molecular, temporal and brain region-specific requirements of CAMK2B for locomotion. At the molecular level we found that normal locomotion requires Calcium/Calmodulin mediated activation of CAMK2B, but CAMK2B autonomous activity is largely dispensable. At a systems level, we found that global deletion of Camk2b in the adult mouse causes only mild locomotion deficits, suggesting that the severe locomotion deficits of Camk2b–/– mice are largely of developmental origin. However, early onset deletion of Camk2b in cerebellum, striatum or forebrain did not recapitulate the locomotion deficits, suggesting that these deficits cannot be attributed to a single brain area. Taken together, these results provide the first insights into the molecular, temporal and region-specific role of CAMK2B in locomotion.

Proopiomelanocortin, glucocorticoid, and CRH receptor expression in human ACTH-secreting pituitary adenomas

ACTH-secreting pituitary tumors are by definition partially autonomous, i.e., secrete ACTH independent of physiological control. However, only few, small-sized studies on proopiomelanocortin (POMC) and its regulation by corticotropin-releasing hormone (CRH) or glucocorticoids are available. Objective of the present study was to report on constitutive and CRH- and dexamethasone-regulated POMC, CRH (CRH-R1), and glucocorticoid receptor (NR3C1) gene expression in a large series of human corticotrope adenomas. Fifty-three ACTH-secreting adenomas were incubated with 10 nM CRH or 10 nM dexamethasone for 24 h. POMC, CRH-R1,NR3C1, and its alpha and beta isoforms were quantified and medium ACTH measured. Constitutive POMC expression proved extremely variable, with macroadenomas exhibiting higher levels than microadenomas. POMC increased during CRH in most specimens; conversely, changes induced by dexamethasone were varied, ranging from decrease to paradoxical increase. No correlation between POMC and ACTH was detected in any experimental condition. CRH-R1 expression was not linked to the response to CRH while NR3C1 was expressed at greater levels in specimens who failed to inhibit during dexamethasone; glucocorticoid receptor α was the more abundant isoform and subject to down-regulation by dexamethasone. Our results demonstrate a considerable variability in POMC expression among tumors and no correlation between POMC and ACTH, suggesting that POMC peptide processing/transport plays a major role in modulating ACTH secretion. Further, CRH-R1 and NR3C1 expression were not linked to the expected ligand-induced outcome, indicating that receptor signaling rather than abundance determines corticotrope responses. Our findings pave the way to new avenues of research into Cushing’s disease pathophysiology.

XAF1 promotes neuroblastoma tumor suppression and is required for KIF1Bβ-mediated apoptosis.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bβ, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bβ deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bβ. XAF1 silencing protects from NGF withdrawal and from KIF1Bβ-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions.

Glucocorticoid Receptor Beta Enhances Myoblast Proliferation and Myotube Formation, and Reduces Dexamethasone‐Induced Atrogene Expression

BackgroundChronic elevations in glucocorticoids from endogenous production or exogenously due to disease treatment, cause skeletal muscle proteolysis and loss of muscle mass. The alpha isoform of the glucocorticoid receptor (GRα) but not the beta isoform (GRβ), has a glucocorticoid binding domain, implicating GRα as the mediator of glucocorticoid‐induced proteolysis. Because GRβ is known to cause glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. To date, there is a significant lack of research on the role of GRβ in the regulation of glucocorticoid‐induced skeletal muscle atrophy.PurposeThe purpose of this study was to determine how the overexpression of GRβ affects myotube formation and the responsiveness of atrogenes to dexamethasone.MethodsMouse GRβ cDNA was overexpressed in C2C12 mouse myoblasts using lentivirus. Changes in cell proliferation were quantified using an MTT assay. Myosin heavy chain staining of day 4 myotubes was used to quantify the effects of GRβ overexpression on indices of myotube formation. Real‐time PCR was used to investigate changes in mRNA expression of glucocorticoid responsive genes and western blot was used to quantify changes in protein expression in response to GRβ overexpression.ResultsGRβ protein expression in C2C12 mouse myoblasts was unaffected by dexamethasone, however, increased in response to insulin. Lentiviral‐mediated GRβ overexpression increased mRNA levels of MyoD and myogenin in confluent myoblast cultures. Furthermore, GRβ overexpression led to an enhanced proliferation of myoblasts associated with reduced mRNA levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Following four days of differentiation, GRβ overexpressing myotubes had increased total number of nuclei within myotubes, nuclei per myotube, the number of myotubes, and a greater fusion index. Lastly, when treated with dexamethasone for 24 hours, GRβ overexpressing myotubes had a blunted MAFbx and MuRF1 atrogene response.ConclusionsOverexpression of GRβ in skeletal muscle may reduce the catabolic effects of glucocorticoids on skeletal muscle by reducing atrogene responsiveness, primarily causing a state of glucocorticoid resistance. Increasing GRβ levels may stabilize muscle mass and preserve metabolic and physical function during exposure to high doses of glucocorticoids. Future work is warranted to determine the effects of GRβ overexpression in vivo in response to physical, social, and pharmacological models of glucocorticoid excess.

The role of PKC‐B in the BKCa functionality of vascular smooth muscle of diabetic rats

Diabetes mellitus (DM) is a disease characterized by high blood glucose concentrations. Cardiovascular disorders associated with DM are the leading cause of high morbidity and mortality of this disease, where endothelial tissue as well as smooth muscle is damaged. For example, the contractility of the smooth muscle cells is increased partly due to a decrease in the activity of the calcium‐dependent high conductance potassium channels (BKCa), which along with other potassium conductance maintain membrane potential and vascular tone.Protein kinase C (PKC) is involved in signaling pathways that promote muscle contraction. Christiandimitropoulou (2002) reported that in the vascular smooth muscle of diabetic rats the outwardly currents flowing through the BKCa channels are decreased. Likewise it was also reported that PKC can phosphorylate some residues of the carboxyl terminal of the alpha subunit of said channel. Data from our laboratories show that the inhibition of the classic isoforms of the PKC is associated with a decrease in the contraction of the mesenteric artery of diabetic rats, which suggests that the beta isoform may be involved in the vascular damage observed in DM.ObjectivesTo study the role of the PKC‐B on BKCa channels functionality and the aortic vascular contractility of diabetic rats induced by streptozotocine.MethodsTension and western blot experiments were performed on aortic rings.Resultsthe NS11021, a BKCa channel activator, significantly relaxes the rings of diabetic rats on a lesser scale than those of the control group, the PKC‐B LY333531 inhibitor does not modify the contractility induced by phenylephrine in normal rings but importantly relaxes those from diabetic rings. The effect of NS11021 is preserved even in the presence of LY333531. Western blot experiments suggest that smooth muscle from diabetic rats is less phosphorylated, indicating that BKCa activity is reduced compared with those from control rats.Conclusionsvascular smooth muscle of diabetic rats show greater contractility and this may be due to a diminished activity of the BKCa channels.Support or Funding InformationSupported by C14‐FAI‐04‐33.33 to PAS, Conacyt 223350‐M to RET. Conacyt scholarship to JAM (295659)

Human alpha3 beta1 isoform of Na+/K+‐ATPase: expression in yeast, mutations and their relation to neurological disorders

The Na+/K+‐ATPase (NKA) is a crucial membrane protein responsible for maintaining the membrane potential by actively transporting sodium and potassium ions. The proper function of this pump is essential for all cells (e. g. in homeostasis, membrane potential). The human body contains four isoforms of this enzyme, which are spread over whole organism. One of the most important localizations of NKA is the brain. Medical research of Heinzen et al. (2014) suggests that there is a possible connection between NKA alpha3 isoform mutations (mainly D801N, E815K, D923N, D923Y and G947R) and neurological disorders. Detailed studies of patient's genome revealed that all these mutations are closely linked to Rapid‐Onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood.Modern biotechnological methods, especially the production of a heterologous protein in expression systems, enable deeper study of a wide range of non‐abundant proteins. The preparation of alpha3 NKA proteins (wild type and mutants) was performed according to Pedersen et al. (2006) with some modifications. In general, changes in the level of protein expression and determination of the relative enzyme activity could be the first step towards better understanding of the relation between NKA function and neurological disorders.Support or Funding InformationThis project was supported by grant No. L01204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS and by “Palacky University Foundation 2015”. The project was awarded by EMBO Short‐Term fellowship ASTF 243‐2015.

A PHASE 1 RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, MULTIPLE ASCENDING DOSE STUDY TO EVALUATE SAFETY, TOLERABILITY AND PHARMACOKINETICS OF THE LIVER-SELECTIVE TR-BETA AGONIST VK2809 (MB07811) IN HYPERCHOLESTEROLEMIC SUBJECTS

Thyroid hormone is a key regulator of lipoprotein metabolism, particularly acting via the beta isoform of nuclear T3 receptor in liver. VK2809 is a small molecule prodrug of a potent thyroid beta agonist. VK2809 is selectively cleaved in hepatic tissue by the action of cytochrome P450 isozyme 3A4,

Transforming growth factor beta (TGF-β) isoforms in wound healing and fibrosis.

Scar formation, with persistent alteration of the normal tissue structure, is an undesirable and significant result of both wound healing and fibrosing disorders. There are few strategies to prevent or to treat scarring. The transforming growth factor beta (TGF-β) superfamily is an important mediator of tissue repair. Each TGF-β isoform may exert a different effect on wound healing, which may be context-dependent. In particular, TGF-β1 may mediate fibrosis in adults' wounds, while TGF-β3 may promote scarless healing in the fetus and reduced scarring in adults. Thus, TGF-β3 may offer a scar-reducing therapy for acute and chronic wounds and fibrosing disorders.

Abstract OT1-03-06: LORELEI: A phase II randomized, double-blind study of neoadjuvant letrozole plus taselisib (GDC-0032) versus letrozole plus placebo in postmenopausal women with ER-positive/ HER2-negative, early-stage breast cancer

Abstract Background: Taselisib is an orally bioavailable, potent, selective inhibitor of Class I PI3-kinase (PI3K) alpha, gamma, and delta isoforms, with 30-fold less inhibition of the PI3K beta isoform relative to the alpha isoform showing enhanced activity against PIK3CA mutant cancer cell lines. Clinical data have demonstrated confirmed partial responses in patients with PIK3CA mutant breast cancer (BC) treated with single-agent taselisib. Enhanced antitumor activity has been noted when taselisib is combined with either letrozole or fulvestrant in preclinical and Phase Ib clinical studies. Methods: LORELEI is a Phase II, two-arm, randomized, double-blind, multicenter, study of neoadjuvant letrozole and taselisib versus letrozole and placebo in postmenopausal women with newly diagnosed ER+/HER2-, untreated, Stage I-III operable BC. Other eligibility criteria include tumor size  2 cm by magnetic resonance imaging (MRI), ECOG PS 0-1, and evaluable tumor tissue for PIK3CA genotyping. Patients treated with anti-diabetic drugs are not eligible. Patients are randomized (1:1) to receive continuous letrozole (2.5 mg) with either taselisib (4 mg on a 5 days on/ 2 days off schedule) or placebo for 16 weeks, followed by surgery. Stratification is based on tumor size and nodal status. The co-primary endpoints are overall objective response rate (ORR) by centrally assessed breast MRI via modified RECIST criteria and pathologic complete response (pCR) rate in breast and axilla at time of surgery in all randomized patients and PIK3CA mutant patients. Secondary endpoints include ORR by centrally-assessed MRI and pCR rate in PIK3CA wild-type patients. The sample size was calculated to detect an absolute percentage increase of 24% in ORR with 80% power and an absolute percentage increase of 18% in pCR rate. An interim safety analysis will be conducted by an Independent Data Monitoring Committee. As of 1st Jun 2015, 54 of the 330 patients have been enrolled, and global enrollment is ongoing (clinicaltrials.gov NCT02273973). Contact information: Reference Study ID Numbers: GO28888/BIG-3-13/SOLTI 1205/ABCSG 38 Phone: 888-662-6728 (US Only) Email Address: global.rochegenentechtrials@roche.com Citation Format: Oliveira M, de Azambuja E, Saura C, Dubsky P, Zardavas D, Fesl C, Bardia A, Soberino J, Ciruelos Gil E, Ng V, Fredrickson J, Stout TJ, Singel SM, Hsu JY, Piccart M, Gnant M, Baselga J. LORELEI: A phase II randomized, double-blind study of neoadjuvant letrozole plus taselisib (GDC-0032) versus letrozole plus placebo in postmenopausal women with ER-positive/ HER2-negative, early-stage breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-03-06.

Biochemical Comparison of Rabbit Skeletal Muscle Alpha and Beta Tropomyosin Isoforms

The alpha and beta isoforms of tropomyosin (39 amino acid substitutions) were isolated from adult rabbit skeletal muscle without chemical modification. The levels of phosphorylation as judged by 2D PAGE were ∼10% (alpha) and nil (beta). Solutions of homodimeric beta tropomyosin (pH 7; ionic strength, ∼30 mM) display greater viscosity than those of alpha. Conversely, the enhancement of viscosity of the alpha isoform by skeletal troponin is greater than for beta. Mixtures of tropomyosin and the amino-terminal CNBr fragment of troponin-T, CB1 (res 1 - 151), were chromatographed on a size-exclusion column in the presence of different concentrations of KCl. In 0.1M salt, CB1 complexes and co-elutes with each isoform but is dissociated from either in 0.22 M. At intermediate salt, different degrees of complexation are observed - more extensive with alpha than beta. Further, the alpha-isoform elutes later in the salt gradient than the beta isoform during chromatography on skeletal troponin-Sepharose 4B. Tropomyosin isotype also has a bearing on thin filament regulation of the steady-state rate of actomyosin-S1 MgATPase. Reconstituted skeletal thin filaments composed solely of beta produce ∼30% less Ca(II)-induced activation compared to only alpha. This is observed at a high S1/actin ratio (6 uM myosin-S1A1, 3 uM thin filaments, pCa 4) and as a function of pCa (0.3 uM myosin-S1A1, 25 uM thin filaments).

The correlation between PLC-β2 and miR-146a in breast ductal carcinoma in situ (DCIS) defines its malignant potential

Ductal carcinoma in situ (DCIS), which represents the most frequently diagnosed tumor in women in industrialized countries, may be a crucial step in the progression of breast lesions to invasive ductal carcinoma (IDC) (1). Among the signaling molecules deregulated in breast tumors, the beta2 isoform of the phosphoinositide-dependent phospholipase C (PLC-b2) strongly correlates with malignancy of invasive tumors and breast tumor-derived cells (2, 3). In breast tumor-derived cell lines cultured under hypoxia, PLC-b2 regulates the levels of cancer stem cell and epithelial-to-mesenchymal transition (EMT) markers (4), suggesting its involvement in breast cancer progression. By using archival FFPE breast tumor samples, we demonstrated that PLC-β2 is up-regulated in DCIS, in which it inversely correlates to the levels of miR-146a, known to act as a tumor suppressor in breast cancer (5). By using the MCF10DCIS cell line, a well-established model of DCIS-derived cells, we demonstrated that the de-regulation of miR-146a is sufficient to modulate the expression of PLC-β2, in turn able to affect the epithelial-to-mesenchymal shift as well as the number of cells expressing CD133. These data indicate that miR-146a and PLC-β2 are members of an intracellular network able to ensure the maintenance of the non-invasive phenotype of DCIS and suggest that alterations in their levels can determine the appearance of an invasive phenotype. The potential prognostic relevance of PLC-β2/miR-146a relationship was investigated in primary DCIS from patients who developed an invasive ductal carcinoma in the contralateral breast. The PLC-β2/miR-146a correlation was found negative in primary DCIS from patients who did not recur and strongly positive in DCIS from patients who developed a contralateral IDC. We propose that the assessment of the correlation between the levels of PLC-β2 and miR-146a in primary DCIS at diagnosis could be beneficial to identify patients with either low or high propensity to develop invasive recurrence. Since a major problem in the management of patients with DCIS is the lack of reliable prognostic markers, our results might be of value in selecting the most appropriate therapies for individual women with non-invasive breast neoplasia.

Preparation of some metabolites of timosaponin BII by biotransformation in vitro

Timosaponin BII (TBII) is an active constituent of some Chinese medicinal herbs, and is abundant in Anemarrhena asphodeloides Bunge. To better understand the pharmaceutical mechanisms of TBII, we prepared its deglycosylated derivatives through biotransformation with fungi Colletotrichum gloeosporioides, Acremonium alternatum and Aspergillus niger. In the presence of 10g/L glucose, transformation with C. gloeosporioides yielded 4 products: timosaponin AIII (P3) and AI (P5) and their beta isoforms (P2 and P4 respectively). However, P4 and P5 could not be detected when 0–5g/L glucose was applied; P2 and P3 disappeared at 20g/L glucose. P4 is a newly discovered compound. We firstly report that fungi in whole cell model can hydrolyze C3-O-glucopyranosyl residues of steroidal saponins after removing the C26 sugar. Therefore, the transformation efficiency depends on glucose. Transformation of TBII, and maybe other steroidal saponins, with C. gloeosporioides is an ideal method to prepare the metabolites in vitro.

Age-Related Changes of 14-3-3 Isoforms in Midbrain of A53T-SNCA Overexpressing Mice.

Parkinson's disease (PD) is characterized by loss of midbrain dopaminergic neurons, which are affected by cytoplasmic inclusions, named Lewy pathology. The main component is alpha-synuclein (SNCA), a protein modulating SNARE-complex dependent neurotransmission. SNCA mutations trigger dominantly inherited PD variants and sporadic cases of PD via aggregation and transmission. SNCA and isoforms of the 14-3-3 family show sequence homology, protein interaction and joint aggregation, so 14-3-3 s may be key molecules of pathogenesis. We aimed to identify the relevant isoforms in midbrain and to distinguish for the first time the changes that occur very early versus those that progress with pathology. We assessed expression of the 14-3-3 family with quantitative RT-PCR and immunoblots of differential solubility fractions in mice with A53T-SNCA overexpression longitudinally at different ages. Transcript levels showed reductions at age 3 months with increases at later ages for the beta, eta and zeta isoforms. Protein levels at age 3 months exhibited a concordant reduction only for beta, while increased insolubility was observed for epsilon and zeta. At age 18 months only the reduction of 14-3-3 beta protein remained significant. Thus, the toxic gain-of-function of alpha-synuclein leads to early transitory alterations of several 14-3-3 isoforms. When the levels of soluble 14-3-3 proteins become apparently normal during later life, increasing amounts of beta, eta and zeta mRNA are produced, possibly to compensate for protein insolubility and aggregation in a SNCA/14-3-3 complex. These data may contribute to identify key molecular events that reflect Parkinson's disease risk and progression.

Abstract 2673: Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines

Abstract Diffuse large B-cell lymphoma (DLBCL) is the largest lymphoma subtype representing approximately one-third of all cases of non-Hodgkin's lymphoma. Gene expression profiling showed that DLBCL can be stratified into activated B-cell (ABC) or germinal center B-cell (GCB) subtypes (Alizadeh et al., Nature 2000). The overall five year survival rate is only approximately 50% (Shaffer et al., Annu. Rev. Immunol. 2012), thus a medical need exits for the treatment of this disease, particularly for patients who relapse after the first line chemoimmunotherapy regimen. Idelalisib is an investigational, highly selective oral inhibitor of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) delta isoform. PI3K delta signaling is required for B lymphocyte activation, proliferation and survival and is dysregulated in several B-cell malignancies. We investigated the activity of idelalisib and ibrutinib, an inhibitor of Bruton's tyrosine kinase, on the inhibition of proliferation against a panel of 22 DLBCL (9 ABC, 13 GCB) and 3 transformed follicular lymphoma cell lines. Idelalisib has potent activity (EC50<300nM) against 3 of 9 (33%) ABC, 3 of 13 (23%) GCB DLBCL and 1 of 3 (33%) transformed follicular lymphoma cell lines at clinically achievable concentrations (culture media protein binding adjusted Cmax 421nM). In contrast, ibrutinib has activity (EC50<40nM) against only 3 of 9 (33%) ABC DLBCL cell lines at clinically achievable concentrations (culture media protein binding adjusted Cmax 48nM). Interestingly, the same three ABC DLBCL cells lines are sensitive to both idelalisib and ibrutinib. Although all of the B-lymphoma cell lines express multiple PI3K isoforms, the PI3K delta isoform is the most highly expressed across all malignant B lymphoma cell lines evaluated. We show that PI3K delta is essential for proliferation in the idelalisib sensitive cell lines using PI3K alpha, beta, or delta isoform selective inhibitors. Further, we demonstrate that idelalisib blocks AKT and RPS6 phosphorylation. Activating mutations in CD79 subunits of the B-cell receptor are associated with sensitivity to idelalisib and ibrutinib in ABC DLBCL cell lines. The absence of PTEN protein in GCB DLBCL cell lines does not correlate with increased sensitivity to idelalisib. Baseline and drug treatment gene expression analysis to investigate differential pathways driving growth and survival in responding versus non responding cell types has been performed and results will be presented. These data support advancing idelalisib in clinical trials for the treatment of a select subgroup of DLBCL patient population Citation Format: Jia Y. Liu, Tom Kenney, Leslie Butterworth, Adam Kashishian, Sarah Meadows, Peng Yue, Li Li, Kathleen Keegan, Christophe Quéva, Stacey Tannheimer. Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2673. doi:10.1158/1538-7445.AM2015-2673

Dual inhibition of classical protein kinase c-alpha and beta isoforms reduces epithelial-to-mesenchymal transition of mesothelial cells and prevents peritoneal damage in a mouse model of chronic peritoneal exposure to high glucose dialysate

Dual inhibition of classical protein kinase c-alpha and beta isoforms reduces epithelial-to-mesenchymal transition of mesothelial cells and prevents peritoneal damage in a mouse model of chronic peritoneal exposure to high glucose dialysate

Evidence for changes in beta- and gamma-actin proportions during inner ear hair cell life.

Cytoplasmic actin isoforms beta (β-) and gamma (γ-) perform crucial physiological roles in inner ear hair cells (HC). The stereocilium, which is structured by parallel actin filaments composed of both isoforms, is the responsive organelle to mechanical stimuli such as sound, gravity and head movements. Modifications in isoform proportions affect the function of the stereocilia as previously shown in genetic studies of mutant mice. Here, immunogold labeling TEM studies in mice showed that both β- and γ-actin isoforms colocalize throughout stereocilia actin filaments, adherens junctions and cuticular plates as early as embryonic stage 16.5. Gold-particle quantification indicated that there was 40% more γ- actin than β-actin at E16.5. In contrast, β- and γ-actin were equally concentrated in adult stereocilia of cochlear and vestibular HC. Interestingly, all actin-based structures presented almost five-fold more β-actin than γ-actin in 22 month- old mice, suggesting that γ-actin is probably under-expressed during the aging process. These data provide evidence of dynamic modifications of the actin isoforms in stereocilia, cuticular plates and cell junctions during the whole HC life.