Isoform-specific Knockdown Research Articles

NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis.

The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.

Isoform-specific knockdown of PKC isoforms reveals PKC[beta]2 is required for epidermal growth factor stimulated expression of the prostaglandin synthase COX2 in human myometrium

Isoform-specific knockdown of PKC isoforms reveals PKC[beta]2 is required for epidermal growth factor stimulated expression of the prostaglandin synthase COX2 in human myometrium

Abstract A60: The hMENA Splicing Program: An important regulator of TGFβ1-driven EMT and invasiveness in pancreatic cancer

Abstract Background: The pancreatic ductal adenocarcinoma tumor microenvironment plays an important role in promoting the epithelial to mesenchymal transition (EMT), an early event in pancreatic cancer, involved in cancer invasiveness and in tumor progression. Among the stromal components the cancer-associated fibroblasts (CAFs) are responsible for the peculiar pancreatic tumor microenvironment and are known to be linked to the induction of EMT. The EMT process requires a dynamic remodeling of the actin cytoskeleton and we have suggested that the splicing program of hMENA, an actin regulator, play a role in EMT. Two alternatively expressed isoforms, hMENA11a and hMENAΔv6, with opposite functions in invasiveness have been described in breast cancer (Di Modugno et al PNAS 2012). hMENA expression has not been detected in normal pancreatic ducts, whereas expressed in the human pancreatic ductal adenocarcinoma (PDAC) samples, but no data are available on hMENA alternative isoform expression in this neoplasia. The aim of this study is to investigate whether TGFβ1-mediated EMT in pancreatic cancer cells is affected by hMENA overexpression and splicing and how CAFs affect this process in cancer cell lines and in human tissues. Methods: hMENA isoform expression was evaluated in PDAC tissues by immunohistochemistry using isoform specific antibodies. hMENA isoforms and EMT markers expression were characterized in human PDAC cell lines, TGFβ1-treated or untreated, by qRT-PCR and WB analysis. The effects of either hMENA isoform specific knockdown or overexpression in the TGFβ1-induced EMT were also evaluated. Pancreatic CAFs were isolated from human tissues of resected PDAC patients. The effect of the conditioned medium of cultured CAFs was evaluated on hMENA expression. In parallel, the role of CAF-cancer cell interaction on the expression of the different hMENA isoforms was analysed using a co-culture system. Results: Freshly explanted CAFs expressed the “mesenchymal” hMENAΔv6, and not hMENA11a and secreted paracrine factors involved in the induction of hMENA isoforms in tumor cells. In a panel of pancreatic cancer cell lines, hMENA11a expression correlated with an epithelial phenotype, while hMENAΔv6 expression was correlated with a mesenchymal phenotype. Interestingly, the expression of the invasive hMENAΔv6 isoform is specifically up-regulated by TGFβ1 treatment. hMENA isoform expression levels influenced molecular changes induced by TGFβ1. Thus, the hMENA11a specific silencing led to E-cadherin down-regulation that is more evident in TGFβ1 treated cells. On the contrary, hMENA11a overexpression led to a reduction of vimentin expression and to E-cadherin up-regulation. Knockdown of the endogenous hMENA/hMENAΔv6 isoform expression prevented the activation of TGFβ1 signaling and up-regulation of mesenchymal markers. In addition, hMENA/hMENAΔv6 isoform depletion impaired the TGFβ1-induced invasiveness, migration and production of MMPs. IHC analysis of PDAC tissues revealed that the epithelial hMENA11a is rarely expressed in primary pancreatic tumour, while high levels of hMENA and hMENAΔv6 isoforms were found in 75% of primary tumours analysed. Conclusions: This data suggests that the lack of the epithelial hMENA11a isoform is an early event in pancreatic cancer, provides new insights into the role of hMENA splicing in TGFβ1-mediated EMT and highlights hMENA splicing program as an attractive pathway for the development of new therapies in PDAC. Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Novella Gualtieri, Maria Grazia Diodoro, Marcella Mottolese, Gian Luca Grazi, Matteo Fassan, Aldo Scarpa, Mina J. Bissell, Paola Nisticò. The hMENA Splicing Program: An important regulator of TGFβ1-driven EMT and invasiveness in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A60.

AKT1 and AKT2 induce distinct phosphorylation patterns in HL-1 cardiac myocytes.

The protein kinase AKT is a central kinase in the heart and has a major impact on growth/hypertrophy, survival/apoptosis, and metabolism. To gain more insight into AKT isoform-specific signaling at the molecular level, we investigated the phosphoproteome of HL-1 cardiomyocytes carrying AKT1 or AKT2 isoform-specific knock down, respectively. We combined stable isotope labeling with high resolution mass spectrometry and identified 377 regulated phosphopeptides. Although AKT1 is expressed at 4-fold higher levels, insulin stimulation mainly activated AKT2, which might in part rely on a preferred interaction of AKT2 with the mammalian target of rapamycin complex 2. In line with this result, the highest number of regulated phosphopeptides was identified in the AKT2 knock down cells. Isoform-specific regulation of AKT targets not previously described could be observed, and specific regulation of indirect target sites allows a deeper insight into affected biological processes. In the myocardial context, we identified many phosphosites supporting a connection of AKT to excitation-contraction coupling. Phosphoproteins identified included L-type calcium channel, ryanodine receptor, junctophilin, histidine-rich calcium binding protein, phospholamban, heat shock protein beta-6, and Ca²⁺/calmodulin-dependent kinase II. In conclusion, AKT isoform-specific knock down combined with quantitative phosphoproteomics provided a powerful strategy to unravel AKT isoform-specific signaling.

PI3K-dependent multiple myeloma cell survival is mediated by the PIK3CA isoform.

Constitutive phosphatidylinositide 3-kinase (PI3K) signalling has been implicated in multiple myeloma (MM) pathophysiology and is regarded as an actionable target for pharmacological intervention. Isoform-specific PI3K inhibition may offer the most focused treatment approach and could result in greater clinical efficacy and reduced side effects. We therefore performed isoform-specific knockdown of PIK3CA, PIK3CB, PIK3CD, and PIK3CG to analyse their individual contributions to MM cell survival and downstream signalling. In addition, we tested the effectivity of the novel PI3K isoform-specific inhibitors BYL-719 (PIK3CA), TGX-221 (PIK3CB), CAL-101 (PIK3CD), and CAY10505 (PIK3CG). We found the PIK3CA isoform to be of paramount importance for constitutive Akt activity in MM cells, and - in contrast to inhibition of other class I isoforms - only the blockade of PIK3CA was sufficient to induce cell death in a sizeable subgroup of MM samples. Furthermore, pharmacological PIK3CA inhibition in combination treatments of BYL-719 and established anti-myeloma agents resulted in strongly enhanced MM cell death. Our data thus clearly indicate therapeutic potential of PIK3CA inhibitors and support their clinical evaluation in multiple myeloma.

Class I PI3K Isoforms Exert a Differential Role On Survival and Cell Trafficking In Multiple Myeloma

BackgroundThe phosphatidylinositol-3 kinase (PI3K) pathway is a critical regulator of tumor progression, protein translation and cytoskeletal dynamics, collectively required for cell proliferation, survival, adhesion and migration in many malignancies including multiple myeloma (MM). Despite the absence of mutations in the PI3K/Akt genes, many studies have demonstrated that this pathway is constitutively activated in MM cells. In this study, we investigated the role of inhibition of class I PI3K isoforms known as p110α, p110β, p110γ and p110δ in cell trafficking of MM cells using isoform-specific knockdown (KD). We have also evaluated the effect of pan-PI3K inhibitior, NVP-BKM120, on survival, adhesion and migration of MM cells both in vitro and in vivo. MethodsThe baseline expression of class I PI3K isoforms in MM cell lines (MM.1S, OPM1, OPM2, H929, RPMI, INA6, U266, and U266LR7) has been evaluated by immnunobloting. MM tumor cells (MM.1S-GFP+/luc+) were infected with lentivirus mediated shRNA targeting class I PI3K isoforms. RT-qPCR and immunoblotting were performed to show infection efficiency. In vivo tumor growth of isoform specific KDs were assessed by using in vivo bioluminescence (BLI) in SCID mice. Detection of circulating MM-GFP+ cells ex vivo was performed by flow cytometry. Analysis of circulating tumor cells for each isoform-specific KD cells against relative tumor volume was performed by lineer regression using GraphPad software. Survival, adhesion and migration of KD cells were tested by MTT, adhesion and migration in vitro assay, respectively. NVP-BKM120, a pan-PI3K-inhibitor (Novartis, MA) has been tested both in vitro and in vivo. Ex vivo detection of mobilization and tumor growth of MM cells (MM.1S-GFP+/luc+) treated with 1) vehicle; 2) NVP-BKM120 in SCID mice were assessed by using flow cytometry and in vivo BLI. Homing of MM cells to the BM of mice pre-treated with NVP-BKM120 was evaluated by in vivo confocal. Increased concentrations of NVP-BKM120 have been tested on survival, cell cycle and apoptotic pathways in MM cells, by using MTT, PI staining in flow cytometry and immunoblotting, respectively. NVP-BKM120 induced dose-dependent effect on chemotaxis and adhesion of MM.1s to BM stromal cells (BMSCs) and fibronectin were tested by migration and adhesion assays. ResultsPI3K-p110β was highly expressed in all cell lines; while other isoforms were expressed in some of the MM cell lines tested. Of note, MM.1S expressed all isoforms. Mice injected with PI3K isoform specific knockdown MM.1S cells presented with different tumor burdens; p110β and p110δ mice showed significantly slower tumor progression compared to scramble control cell line (P<.05), whereas tumor growth was similar in p110α and p110γ to control mice. We next compared the number of circulating tumor cells (CTCs) at the same tumor burden between groups, which showed only p110β presented with a higher number of CTCs compared to the scramble group (P=0.01). In vitro, we observed reduced adhesion and enhanced migration of KD cells compared to control with no cell survival difference. The effect of pan-inhibition of PI3K with NVP-BKM120 induced MM cell mobilization from the BM to the circulation (Vehicle: 0.002 % vs NVP-BKM120: 0.023%; P<.05). This was supported by the inhibition of homing of MM cells to the BM (84% decrease) in the mice pre-treated with NVP-BKM120 (P<.05). Furthermore, treatment of mice with 50mg/kg of NVP-BKM120 once a day by oral gavage for five weeks significantly decreased the rate of tumor progression in MM compared to the vehicle treated group, as shown by BLI (69% decrease, P<.01). NVP-BKM120 decreased the activation of adhesion-related signaling in MM cells induced by co-culture with stroma, including pFAK, pSrc, pCoffilin and pMLC, as shown by immunoblotting. Moreover, it caused cell cycle arrest, as detected by PI staining and analyzed by flow cytometry. ConclusionThis study suggests that inhibition of Class I PI3K isoforms, particularly p110β and p110δ, can play an important role in the regulation of cell trafficking in MM by disrupting adhesion of MM cells to the BM and inducing mobilization. Thus, pan-PI3K inhibition by NVP-BKM120 is a promising approach, which may enhance therapeutic response and overcome resistance in the treatment of MM. Disclosures:Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Abstract 267: Opposing Functions of Two Splice Variants of the Human Transcription Factor Grainyhead-like 3 in Endothelial Cells And in vivo

Grainyhead-like 3 (GRHL3) is a member of the evolutionary conserved Grainyhead family of transcription factors. In humans, three isoforms are derived from differential first exon usage and alternative splicing, which differ only in their N-terminus. Isoform 2, the only variant also present in mouse, is required for endothelial cell (EC) migration and protects against apoptosis. The functions of the human specific isoforms 1 and 3, which are derived from an alternatively spliced pre-mRNA, have not yet been investigated, although all three isoforms are expressed in EC. Therefore, we have assessed their effects on EC migration and apoptosis. Overexpression of the two proteins had opposite effects on EC migration, with isoform 1 acting pro-migratory. This protein also protected EC against apoptosis in an eNOS-dependent manner, whereas isoform 3 had no effect. These opposing outcomes with respect to apoptosis EC were corroborated by isoform-specific knockdowns. With reporter assays using a GRHL3-specific luciferase reporter we demonstrated that both are active transcription factors. Microarray analyses revealed that they induce divergent target gene sets in EC. Two validated targets, Akt2 and Mxi1, which are upregulated by isoform1, are regulators of Akt1-, and thus eNOS-phosphorylation and apoptosis, which could explain the effects of this protein on these processes. In vivo, overexpression of isoform 3 in zebrafish embryos resulted in increased lethality and severe deformations, while isoform 1 had no deleterious effect. In conclusion, our data demonstrate that the splice variant derived isoforms 1 and 3 of the human transcription factor GRHL3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

Two Isoforms of Sister-of-Mammalian Grainyhead Have Opposing Functions in Endothelial Cells and In Vivo

Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells

BackgroundDue to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients.MethodsThe activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells.ResultsAKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient.ConclusionOur results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

P3-01-02: EC 304: A Novel Progesterone Receptor (PR) Antagonist Inhibits Transcription and Induces Cell Cycle Arrest in T47D Breast Cancer Cells.

Abstract Progesterone receptor (PR) is a ligand activated transcription factor which plays a crucial role in female reproduction. PR antagonists have been shown to repress estrogen dependent proliferation in the uterus and mammary gland. Even though PR antagonists are potent therapeutic candidates for breast cancer, partial agonism towards glucocorticoid receptor (GR) and androgen receptor (AR) lead to undesirable side effects. Evestra's rational design and development approach in the synthesis of EC304 was aimed at minimizing these side effects as well as to deliver more potent and selective antiprogestins. We used select screen® (Invitrogen) to determine relative binding affinity of EC304 with AR,GR and PR. The binding affinity of EC304 showed strong antagonism towards PR with mild or negligible agonism with AR or GR. PR transactivation was inhibited by EC304 with IC50 of 0.04nM in the presence of progesterone (5nM). EC304 showed superior cytotoxicity over known antiprogestins ZK230211, ORG33628, CDB2914, CDB4124 and RU486 in T47D cells. IC50 of EC304 was found to be 0.5nM in a 6-day cytotoxicity assay (MTT) in T47D cells stimulated with 1nM estrogen (E2). In a 21-day in vitro tumorigenicity assay, EC304 inhibited 80 and 100% of colony formation of T47D cells in the presence of E2 at 1 and 10 nM respectively. Further studies revealed that EC304 induced apoptosis and G1-phase arrest in cell cycle progression when co-incubated with E2 for 24 h. Treatment with EC304 down regulated PR target genes and up regulated cell cycle dependent kinase (CDK) inhibitor P21 in vitro. Ongoing studies address whether EC304 is specifically acting on PR-A or PR-B isoform at gene expression, protein levels and PR isoform specific knockdowns in T47D and other metastatic breast cancer cells. Considered together, these data indicate that EC304 might be a safer but efficacious novel antiprogestin for hormone sensitive postmenopausal breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-02.

FOSL1 Is Integral to Establishing the Maternal-Fetal Interface

Remodeling of uterine spiral arteries by trophoblast cells is a requisite process for hemochorial placentation and successful pregnancy. The rat exhibits deep intrauterine trophoblast invasion and accompanying trophoblast-directed vascular modification. The involvement of phosphatidylinositol 3 kinase (PI3K), AKT, and Fos-like antigen 1 (FOSL1) in regulating invasive trophoblast and hemochorial placentation was investigated using Rcho-1 trophoblast stem cells and rat models. Disruption of PI3K/AKT with small-molecule inhibitors interfered with the differentiation-dependent elaboration of a signature invasive-vascular remodeling trophoblast gene expression profile and trophoblast invasion. AKT isoform-specific knockdown also affected the signature invasive-vascular remodeling trophoblast gene expression profile. Nuclear FOSL1 increased during trophoblast cell differentiation in a PI3K/AKT-dependent manner. Knockdown of FOSL1 disrupted the expression of a subset of genes associated with the invasive-vascular remodeling trophoblast phenotype, including the matrix metallopeptidase 9 gene (Mmp9). FOSL1 was shown to occupy regions of the Mmp9 promoter in trophoblast cells critical for the regulation of Mmp9 gene expression. Inhibition of FOSL1 expression also abrogated trophoblast invasion, as assessed in vitro and following in vivo trophoblast-specific lentivirally delivered FOSL1 short hairpin RNA (shRNA). In summary, FOSL1 is a key downstream effector of the PI3K/AKT signaling pathway responsible for development of trophoblast lineages integral to establishing the maternal-fetal interface.

Splicing factor hnRNPH drives an oncogenic splicing switch in gliomas.

In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-α/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur d'Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16- or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness.

RREB1 Transcription Factor Splice Variants in Urologic Cancer

RREB1 is an alternatively spliced transcription factor implicated in Ras signaling and cancer. Little is known about the expression of RREB1 isoforms in cell lines or human tumors, or about the clinical relevance of the latter. We have developed tools for IHC of RREB1 protein isoform-specific amplification of RREB1 mRNA and selective knockdown of RREB1 isoforms and use these to provide new information by characterizing RREB1 expression in bladder and prostate cancer cell lines and human tissue samples. Previously described splice variants RREB1α, RREB1β, RREB1γ, and RREB1δ were identified, as well as the novel variant RREB1ε. Total and isoform-specific mRNA expression was lower in most but not all tumors, compared with normal tissues. RREB1 IHC performed on a bladder cancer TMA did not indicate a relationship between total RREB1 expression and overall survival after radical cystectomy for invasive bladder cancer. In contrast, in vitro proliferation studies using the UMUC-3 bladder cancer cell line after selective isoform-specific knockdown of expression indicate that RREB1α is not necessary for proliferation, but that RREB1β may be required. These contributions should accelerate progress in the nascent RREB1 field by providing new reagents while also providing clues to the role of RREB1 isoforms in human cancer and raising the possibility of isoform-specific roles in human carcinogenesis and progression.

Targeting individual calpain isoforms for neuroprotection

Targeting individual calpain isoforms for neuroprotection

Abstract 21: Selective and potent inhibitors of the mutant B-Raf pathway paradoxically stimulate the MAPK pathway in wild-type B-Raf cells

Abstract B-Raf is a member of the Raf family of serine/threonine kinases, which also include A-Raf and C-Raf. While all 3 Raf kinases stimulate the MAPK signaling cascade, B-Raf is the most catalytically active. B-Raf hyperactivity through mutation or over-expression is reported in many solid and hematologic malignancies. An especially high frequency (60%) of activating B-Raf mutations is found in melanoma, with the kinase domain mutant (V600E) accounting for greater than 95% of these. The identification and development of B-Raf inhibitors that selectively block signaling of the V600EB-Raf dependent MAPK pathway may have therapeutic value in these and other malignancies driven by activation of B-Raf. We report here on the characterization of novel, potent and selective small molecule Raf kinase inhibitors demonstrating potent cellular activity. Compound 1 (Smith, DeMorin et al. 2009) exhibits excellent potency in cells harboring V600EB-Raf (P-ERK IC50 ranging from 1-14nM) however, cells that contain wild type B-Raf are significantly less sensitive to inhibition of MAPK signaling by compound 1 (P-ERK IC50 ranging from 260nM to &amp;gt; 1μM). This shift in potency between V600EB-Raf versus wtB-Raf cell lines is also observed with cellular viability. In contrast to cells harboring B-Raf mutation, exposure to selected Raf inhibitors resulted in a dose-dependent, and sustained activation of MAPK signaling, increased Raf basal kinase activity and heterodimer stabilization in all wild type B-Raf cell lines examined . In the mutant KRAS MIA PaCa-2 and A549, Raf inhibition by selected compounds led to entry into the cell cycle, 80% increased proliferation, 40% increased colony formation and significantly stimulated tumor growth in vivo (Beltran et al, AACR 2010). Inhibition with structurally distinct Raf inhibitors or isoform specific siRNA knock-down of Raf demonstrated that these effects were mediated directly through Raf. Either A-Raf or C-Raf, mediated the Raf-inhibitor-induced MAPK pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in both malignant and normal cells such as HUVEC cells. Indeed mouse treatment with any of the efficacious inhibitors resulted in hyperplasia observed in the epithelial layer of mouse esophagus and stomach. These data suggest that mutant versus wild type B-Raf MAPK pathways are distinctly regulated in cells. An implication of these results is that certain Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients. Smith, A. L., F. F. DeMorin, et al. (2009). “Selective Inhibitors of the Mutant B-Raf Pathway: Discovery of a Potent and Orally Bioavailable Aminoisoquinoline.” Journal of Medicinal Chemistry 52(20): 6189-6192. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 21.

The Clavesin Family, Neuron-specific Lipid- and Clathrin-binding Sec14 Proteins Regulating Lysosomal Morphology

Clathrin-coated vesicles (CCVs) originating from the trans-Golgi network (TGN) provide a major transport pathway from the secretory system to endosomes/lysosomes. Herein we describe paralogous Sec14 domain-bearing proteins, clavesin 1/CRALBPL and clavesin 2, identified through a proteomic analysis of CCVs. Clavesins are enriched on CCVs and form a complex with clathrin heavy chain (CHC) and adaptor protein-1, major coat components of TGN-derived CCVs. The proteins co-localize with markers of endosomes and the TGN as well as with CHC and adaptor protein-1. A membrane mimic assay using the Sec14 domain of clavesin 1 reveals phosphatidylinositol 3,5-bisphosphate as a specific lipid partner. Phosphatidylinositol 3,5-bisphosphate is localized to late endosomes/lysosomes, and interestingly, isoform-specific knockdown of clavesins in neurons using lentiviral delivery of interfering RNA leads to enlargement of a lysosome-associated membrane protein 1-positive membrane compartment with no obvious influence on the CCV machinery at the TGN. Since clavesins are expressed exclusively in neurons, this new protein family appears to provide a unique neuron-specific regulation of late endosome/lysosome morphology.

Cotranslational and Posttranslational N-Glycosylation of Polypeptides by Distinct Mammalian OST Isoforms

Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation.

Abstract B67: Using alternative splicing microarrays to identify potential biomarkers in lung cancer

Abstract B67 The disruption of alternative splicing (AS) by either mutations in splicing sequences or changes in expression of splicing factors has been linked to many human diseases including cancer but the molecular changes associated with lung tumors are not well understood. Using our established quantitative AS microarray platform we have profiled matched normal and adenocarcinoma tissues from the lungs of 10 patients in order to identify changes at both the AS and transcriptional levels. These profiling experiments show a small set of exons that display pronounced and highly consistent changes between the normal and tumor tissues. This set is distinct from those genes showing changes at the transcriptional level. The results reveal how AS and transcription may be re-programmed during malignant transitions in the lung. Using a custom microarray with sets of exon body and splicing junction probes for profiling ~5000 human cassette alternative exons, we have identified 4 AS events that display pronounced inclusion level differences between normal and adenocarcinoma tissue in at least 80% of patients surveyed. These changes were confirmed by RT-PCR using the original 10 plus an additional 19 patient samples. Interestingly, all 4 of the alternative exons are located in genes that are linked to signaling pathways known to be deregulated in certain cancers. Moreover, these 4 AS events preserve frame and are conserved in mouse tissues, suggesting important functional roles. From the same dataset, a separate set of genes with transcript level changes were detected, consistent with previous findings that non-overlapping sets of genes are regulated at the AS and transcriptional levels, when comparing different tissues or corresponding tissues from different species. In addition to probe sets for profiling AS and transcript levels of the corresponding genes, our microarray contains probes to determine the transcript levels for 465 known and putative splicing factors. While significant changes in the expression levels of defined splicing factors were not detected, we observe changes in expression levels of 3 genes that contain RS domains, a feature of proteins that is predictive of a role in splicing. One of these genes is associated with the Notch pathway and the others are known tumor suppressor genes. We have confirmed that the change in AS for 1 of the target genes results in altered splicing at the protein level in addition to the RNA level changes. Work is in progress to characterize the functional role of this AS event using isoform-specific knockdown and overexpression. The increased expression of the exon-included protein isoform was evident in cell lines derived from both lung and colon cancer. We are currently determining if all 4 AS events occur in tumor tissue from breast and colon cancer patients. Also, we have expanded our profiling of human cancers by using higher density AS microarrays, and by using mRNA samples from additional normal and tumor-derived breast, colon and lung sources. Thus far, we have identified a set of conserved AS events that are consistently associated with lung adenocarcinomas. These are located in genes that function in signaling pathways that play important roles in tumorigenesis. Characterization of these AS events will advance our understanding of the role of splicing in human cancers and may also provide new targets for diagnostic applications as potential biomarkers. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B67.

Alternative Splicing of the Robo3 Axon Guidance Receptor Governs the Midline Switch from Attraction to Repulsion

Alternative splicing provides a means to increase the complexity of gene function in numerous biological processes, including nervous system wiring. Navigating axons switch responses from attraction to repulsion at intermediate targets, allowing them to grow to each intermediate target and then to move on. The mechanisms underlying this switch remain poorly characterized. We previously showed that the Slit receptor Robo3 is required for spinal commissural axons to enter and cross the midline intermediate target. We report here the existence of two functionally antagonistic isoforms of Robo3 with distinct carboxy termini arising from alternative splicing. Robo3.1 is deployed on the precrossing and crossing portions of commissural axons and allows midline crossing by silencing Slit repulsion. Robo3.2 becomes expressed on the postcrossing portion and blocks midline recrossing, favoring Slit repulsion. The tight spatial regulation of opponent splice variants helps ensure high-fidelity transition of axonal responses from attraction to repulsion at the midline.