High Nuclear Accumulation Research Articles

Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity.

Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in cells is missing. To this end, using CRISPR technology, we YFP-tagged the endogenous PSMB6 (β1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with the mScarlet fluorescent protein. We observed the colocalization of the YFP and mScarlet fluorescent proteins in the cells, with higher nuclear accumulation. Nuclear proteasomal granules are formed under osmotic stress, and all were positive for YFP and mScarlet. Previously, we have reported that PSMD1 knockdown, one of the 19 RP subunits, gives rise to a high level of "free" 20S CPs. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet was mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CPs. Lately, we have shown that the PSMA3 (α7) C-terminus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, the truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest that the PSMA3 C-terminal region is critical for 26S proteasome integrity.

Enhanced transfection efficiency and targeted delivery of self-assembling h-R3-dendriplexes in EGFR-overexpressing tumor cells.

The efficient gene transfection, cellular uptake and targeted delivery in vivo are key issues for non-viral gene delivery vectors in cancer therapy. To solve these issues, we designed a new targeted gene delivery system based on epidermal growth factor receptor (EGFR) targeting strategy. An anti-EGFR monoclonal antibody h-R3 was introduced to dendriplexes of PAMAM and DNA via electrostatic interactions to form self-assembled h-R3-PAMAM-DNA complexes (h-R3-dendriplexes). Dendriplexes h-R3-dendriplexes represented excellent DNA encapsulation ability and formed unique nanostructures. Compared to dendriplexes, h-R3-dendriplexes presented lower cytotoxicity, higher gene transfection efficiency, excellent endosome escape ability and high nuclear accumulation in the EGFR-overexpressing HepG2 cells. Both ex vivo fluorescence imaging and confocal results of frozen section revealed that h-R3-dendriplexes showed higher targeted delivery and much better gene expression in the tumors than dendriplexes at the same N/P ratio, and h-R3-dendriplexes had accumulation primarily in the tumor and kidney. Moreover, h-R3-dendriplexes for p53 delivery indicated efficient cell growth inhibition and potentiated paclitaxel-induced cell death. These results indicate that the h-R3-dendriplexes represent a great potential to be used as efficient targeted gene delivery carriers in EGFR-overexpressing tumor cells.

Abstract P2-09-12: Withaferin A-mediated inhibition of breast carcinoma involves concomitant activation of CHOP and Elk1 via ERK/ribosomal S6 kinase (RSK) signaling axis regulating death receptor 5 expression

Abstract Introduction: Despite significant progress towards screening efforts and targeted therapies, breast cancer is still a leading cause of cancer-related mortality among women. Novel therapeutic strategies are clearly needed for effective targeting of breast cancer. Natural products continue to generate interest for identification of potential chemopreventive and therapeutic agents. Withaferin A (WFA) is a bioactive small molecule that has been proposed as a new-generation molecule capable of eliciting growth inhibitory effect on cancer cells. The molecular mechanisms by which WFA mediates inhibition of breast cancer remain elusive. The present study was designed to systematically elucidate the underlying mechanisms by which WFA inhibits growth and metastatic potential of breast cancer cells. Methods: Efficacy of WFA treatment on cell growth was evaluated by using clonogenicity, and anchorage–independent growth. Western blot and immunofluorescence analysis were used to examine activation of ERK/RSK (extracellular signal-regulated kinase/ ribosomal S6 kinase) and downstream signaling axes. Functional importance of ERK/RSK and CHOP (C/EBP homologous protein) in the biologic effects of WFA was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immunohistochemical, RT-PCR and western blot analysis of tumors were used. Results: Analysis of the underlying molecular mechanisms revealed that WFA treatment induced phosphorylation of RSK2 in an ERK-dependent manner. Further, WFA-induced ERK/RSK signaling resulted in concomitant upregulation of CHOP and Elk-1(ETS-like transcription factor 1). By using ERK1/2 siRNA and FMK-MEA inhibitor, we found that ERK/RSK signaling axis is required for WFA-mediated modulation of CHOP and Elk-1. Intriguingly, we discovered that WFA triggers the nuclear translocation of CHOP and Elk1 resulting in transcriptional upregulation of Death Receptor protein (DR5). CHOP and Elk1 overexpression potentiated while phospho-deficient Elk-1 inhibited WFA-induced DR5 expression exhibiting that CHOP/Elk-1 cooperatively regulate DR5 expression. Furthermore, using nontoxic doses of WFA, we showed that WFA treatment effectively inhibited breast tumorigenesis in vivo. Analysis of WFA-treated breast tumors showed increased activation of ERK/RSK axis and DR5 along with higher nuclear accumulation of CHOP and Elk-1. Conclusion: These data provided first in vitro and in vivo evidence of the integral role of a previously unrecognized crosstalk between WFA and ERK/RSK and CHOP/Elk-1 axes in breast tumor growth inhibition. WFA treatment could potentially be a rational therapeutic strategy for breast carcinoma. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-12.

Smaug/SAMD4A Restores Translational Activity of CUGBP1 and Suppresses CUG-Induced Myopathy

We report the identification and characterization of a previously unknown suppressor of myopathy caused by expansion of CUG repeats, the mutation that triggers Myotonic Dystrophy Type 1 (DM1). We screened a collection of genes encoding RNA–binding proteins as candidates to modify DM1 pathogenesis using a well established Drosophila model of the disease. The screen revealed smaug as a powerful modulator of CUG-induced toxicity. Increasing smaug levels prevents muscle wasting and restores muscle function, while reducing its function exacerbates CUG-induced phenotypes. Using human myoblasts, we show physical interactions between human Smaug (SMAUG1/SMAD4A) and CUGBP1. Increased levels of SMAUG1 correct the abnormally high nuclear accumulation of CUGBP1 in myoblasts from DM1 patients. In addition, augmenting SMAUG1 levels leads to a reduction of inactive CUGBP1-eIF2α translational complexes and to a correction of translation of MRG15, a downstream target of CUGBP1. Therefore, Smaug suppresses CUG-mediated muscle wasting at least in part via restoration of translational activity of CUGBP1.

Abstract 5501: Withaferin A-mediated inhibition of breast carcinoma involves concomitant activation of CHOP and Elk1 via ERK/ribosomal S6 kinase (RSK) signaling axis regulating death receptor 5 expression. .

Abstract Introduction: Despite significant progress towards screening efforts and targeted therapies, breast cancer is still a leading cause of cancer-related mortality among women. Novel therapeutic strategies are clearly needed for effective targeting of breast cancer. Natural products continue to generate interest for identification of potential chemopreventive and therapeutic agents. Withaferin A (WFA) is a bioactive small molecule that has been proposed as a new-generation molecule capable of eliciting growth inhibitory effect on cancer cells. The molecular mechanisms by which WFA mediates inhibition of breast cancer remain elusive. The present study was designed to systematically elucidate the underlying mechanisms by which WFA inhibits growth and metastatic potential of breast cancer cells. Methods: Efficacy of WFA treatment on cell growth, invasion and, migration potential was evaluated by using clonogenicity, anchorage-independent growth, matrigel-invasion and spheroid-migration assays. Western blot and immunofluorescence analysis were used to examine activation of ERK/RSK (extracellular signal-regulated kinase/ ribosomal S6 kinase) and downstream signaling axes. Functional importance of ERK/RSK and CHOP (C/EBP homologous protein) in the biologic effects of WFA was examined by using overexpression, phospho-deficient constructs and specific inhibitors. Finally, mouse xenografts, immunohistochemical, RT-PCR and western blot analysis of tumors were used. Results: Analysis of the underlying molecular mechanisms revealed that WFA treatment induced phosphorylation of ERK which in turn activated RSK. Further, WFA-induced ERK/RSK signaling resulted in concomitant upregulation of CHOP and Elk-1(ETS-like transcription factor 1). By using ERK1/2 siRNA and FMK-MEA inhibitor, we found that ERK/RSK signaling axis is required for WFA-mediated modulation of CHOP and Elk-1. Intriguingly, we discovered that WFA triggers the nuclear translocation of CHOP and Elk1 resulting in transcriptional upregulation of Death Receptor protein (DR5). CHOP and Elk1 overexpression potentiated while phospho-deficient Elk-1 inhibited WFA-induced DR5 expression exhibiting that CHOP/Elk-1 cooperatively regulate DR5 expression. Furthermore, using nontoxic doses of WFA, we showed that WFA treatment effectively inhibited breast tumorigenesis in vivo. Analysis of WFA-treated breast tumors showed increased activation of ERK/RSK axis and DR5 along with higher nuclear accumulation of CHOP and Elk-1. Conclusion: These data provided first in vitro and in vivo evidence of the integral role of a previously unrecognized crosstalk between WFA and ERK/RSK and CHOP/Elk-1 axes in breast tumor growth inhibition. WFA treatment could potentially be a rational therapeutic strategy for breast carcinoma. Citation Format: Arumugam Nagalingam, Joseph H BOK, Panjamurthy Kuppusamy, Neeraj Saxena, Dipali Sharma. Withaferin A-mediated inhibition of breast carcinoma involves concomitant activation of CHOP and Elk1 via ERK/ribosomal S6 kinase (RSK) signaling axis regulating death receptor 5 expression. . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5501. doi:10.1158/1538-7445.AM2013-5501

Abstract LB-83: Molecular stratification of medulloblastoma: comparison of histological and genetical methods to detect Wnt activated tumors.

Abstract Introduction: Wnt activation in medulloblastomas is associated with good outcome. Therefore, upfront testing and risk-adapted stratification of the patients will be done in future clinical studies. In a cohort of 188 pediatric medulloblastomas our aim was to identify the optimal methods in standard clinical practice to detect this subgroup. Experimental procedures: Nuclear accumulation of ß-Catenin in tumor cells was analyzed by immunohistochemistry (IHC) of sections from formalin-fixed paraffin-embedded (FFPE) tumor samples with 3 different automated immunostaining systems and subsequent neuropathological evaluation. Genomic DNA of FFPE tumor tissue from each sample was amplified by PCR for SSCP (single-strand conformation polymorphism) analysis and direct Sanger sequencing of CTNNB1 exon 3. Copy number aberrations of chromosome 6 were analyzed by MLPA (multiplex ligation-dependent probe amplification) using the same genomic DNA and were confirmed in a subset of cases by molecular invasion profiling. Results: The different automated immunostaining systems showed similar results even though ß-Catenin staining intensity varied. 22 of 188 samples showed nuclear accumulation in >5% of tumor cells and further 21 samples showed ≤5% ß-Catenin positive nuclei. None of the latter 21 cases had mutations in exon 3 of the CTNNB1 gene, but 18 of the 22 cases with >5% nuclear accumulation did, clearly identifying these 18 cases as Wnt-subgroup medulloblastomas. SSCP analysis only showed DNA bands with aberrant migration in 3 of the 18 mutated samples representing 2 different mutations. With MLPA and molecular invasion profiling the 4 cases with >5% nuclear accumulation but without CTNNB1 mutation also lacked monosomy 6, which is another known marker for Wnt subgroup medulloblastomas. 15 of the 18 mutated cases showed monosomy 6, but 3 had a balanced chromosome 6, indicating that monosomy 6 is present in most but not all Wnt cases. On the other hand, none of the CTNNB1 wildtype tumors showed monosomy 6. Conclusion: Standard neuropathological evaluation of medulloblastoma samples should include IHC of ß-Catenin because tumors with high nuclear accumulation of ß-Catenin most probably belong to the Wnt subgroup of medulloblastomas associated with a good outcome. Still, IHC alone may be insufficient because of low quality of paraffin tissue, technical problems or differences in scoring ß-Catenin positive nuclei. Similar, chromosome 6 aberrations in MLPA/molecular invasion profiling were shown not to be present in all CTNNB1-mutated cases. Therefore, we conclude that sequencing analysis of CTNNB1 exon 3 in combination with ß-Catenin IHC (possibly as pre-screening method) is a feasible, fast and cost-efficient way for the determination of Wnt subgroup medulloblastomas. Citation Format: Tobias Goschzik, Anja zur Mühlen, Glen Kristiansen, Torsten Pietsch. Molecular stratification of medulloblastoma: comparison of histological and genetical methods to detect Wnt activated tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-83. doi:10.1158/1538-7445.AM2013-LB-83

Abstract 349: Leptin-induced epithelial-mesenchymal transition in breast cancer cells requires β-catenin activation via Akt/GSK3-dependent and MTA1/Wnt1-dependent pathways

Abstract Perturbations in the adipocytokines-profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide first molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1) and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of GSK3β-LKB1-Axin complex which leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors show increased expression of Wnt1, pGSK3β and Vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized crosstalk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 349. doi:1538-7445.AM2012-349

Comparing the intracellular fate of components within a noncovalent streptavidin nanoparticle with covalent conjugation

Auger radiotherapy requires adequate tumor delivery and high nuclear accumulation and retention. We hypothesize that the noncovalent nature of a streptavidin/biotin three-component nanoparticle possessing these qualities may be required for dissociation of the radiolabeled oligomer and its accumulation into the cell nucleus. As a test of our hypothesis, the intracellular fate of an antisense oligomer when incubated as the nanoparticle and when incubated while covalently conjugated to the antibody was compared. The three-component noncovalent nanoparticle consisted of streptavidin linking three biotinylated components: a Cy3-labeled anti-RIα antisense phosphorodiamidate morpholino (MORF) oligomer, a tat transfecting peptide and the anti-Her2 herceptin antibody. The covalent constructs included an anti-RIα antisense DNA conjugated to a radiolabeled herceptin and a fluorescent DNA conjugated to native herceptin. Fluorescence microscopy in SK-BR-3 (Her2+) cells was used to evaluate the fate of the fluorescent Cy5.5-DNA and Cy3-MORF, while the subcellular accumulation of the (111)In-labeled herceptin and herceptin-DNA in both SK-BR-3 and MDA-MB-231 (Her2) cells was determined by isolating and counting the nuclear fractions. Previously, we demonstrated that when incubated as the three-component nanoparticle consisting of herceptin and streptavidin and (99m)Tc-labeled antisense MORF, only the MORF accumulated in the nucleus of Her2+ cells. In this investigation, clear evidence was observed of nuclear accumulation of the antisense oligomer within the noncovalent nanoparticle as before, but when incubated as the covalent construct, by both fluorescence microscopy and nuclear counting, no evidence of nuclear accumulation was observed. The weaker noncovalent biotin-streptavidin bond may be essential for adequate delivery of the radiolabeled antisense oligomer to the nucleus of tumor cells.

A-Kinase Anchoring Proteins

The heart is a sophisticated organ that continuously pumps blood to ensure that oxygen and nutrients reach the brain, other organs, and peripheral tissue. The right side of the heart pumps blood to the lungs, where oxygen is taken up and carbon dioxide is removed. The left side of the heart then pumps blood to the rest of the body, where oxygen and nutrients are delivered to tissues. This cycle is sustained by the repeated contraction of cardiomyocytes, specialized muscle cells that are designed to contract and respond rapidly to physiological stimuli. Normally, contraction of atrial and ventricular myocytes is activated by action potentials that originate in the sinoatrial node in the wall of the right atrium, often referred to as the pacemaker of the heart. Coupling of this electric signal to contraction (EC coupling) in cardiac myocytes is initiated with Ca2+ influx through L-type Ca2+ channels, which activates intracellular Ca2+ release via ryanodine receptors located in the sarcoplasmic reticulum (SR) and thus induces a global increase in [Ca2+]i that triggers contraction.1,2 The rate at which the sinoatrial node myocytes fire action potentials is modulated by 2 opposing nervous systems. The sympathetic nervous system uses the catecholamine hormones noradrenaline and adrenaline to increase the force and rate of atrial and ventricular contraction. In contrast, the parasympathetic nervous system releases the neurotransmitter acetylcholine to reduce action potentials from the sinoatrial node. Together, these neural systems ensure that cardiac output is matched to the physiological needs of the organism, a process that is often termed “stimulus-response coupling.” Although numerous signal-transduction cascades are operational in cardiomyocytes, G-protein–coupled receptor–signaling pathways play a prominent role. Agonists such as noradrenaline, adrenaline, angiotensin II, and endothelin-1 promote the interaction of their respective receptors with a G-protein heterotrimer comprising α-, β-, …

β-catenin expression in relation to genetic instability and prognosis in colorectal cancer

In the carcinogenesis of colorectal cancer (CRC) genetic instability and dysfunction of the Wnt-signalling pathway play important roles. Most Wnt-signalling dysfunctions lead to the nuclear accumulation of beta-catenin. The aim of the present study was to investigate whether nuclear accumulation of beta-catenin is associated with prognosis and genetic instability. We used immunohistochemistry to study nuclear beta-catenin expression in 67 CRCs. The expression was evaluated in the entire tumour section as mean values and in tumour budding at the invasive margin. We compared the results with chromosomal and microsatellite instability (CIN vs. MSI), p53 accumulation, and clinicopathological variables including survival. The nuclear accumulation of beta-catenin was significantly associated with abnormal p53 expression and aneuploidy, typically for CIN, whereas no tumour with nuclear beta-catenin expression at the invasive margin displayed MSI. The beta-catenin expression pattern did not correlate significantly with CRC patient prognosis when including all stages. However, in the clinically most interesting prognostic group, Dukes' stage B patients, high nuclear accumulation of beta-catenin was associated with a poor prognosis (p=0.01). Our results suggest that nuclear accumulation of beta-catenin in CRC is related to CIN and may be of prognostic importance. However, larger studies are needed to verify these findings.

Expression and subcellular localization of CDK2 and cdc2 kinases and their common partner cyclin A in thyroid epithelial cells: Comparison of cyclic AMP‐dependent and ‐independent cell cycles

Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and GO-S prereplicative phase progression by cyclic AMP as a second messenger for TSH. In its early steps, this mitogenic control is quite distinct from cyclic AMP-independent mitogenic cascades elicited by growth factors. We demonstrate here that TSH (cyclic AMP) and EGF + serum (cyclic AMP-independent) stimulations cooperate and finally converge on proteins that control the cell cycle machinery. This convergence included a common induction of the expression of cyclin A and p34cdc2, and to a lesser extent of p33/38cdk2, which was already expressed in quiescent thyroid cells, and common changes of cdc2 and CDK2 phosphorylations as evidenced by electrophoretic mobility shifts. Kinetic differences in these processes after stimulation by TSH or EGF + serum or by these factors in combination correlated with differences in cell cycle kinetics. Moreover, an immunofluorescence analysis of these proteins using the double labeling of PCNA as a marker of each cell cycle phase shows: (1) a previously undescribed nuclear translocation of CDK2 before S phase initiation; (2) a sudden increase of cdc2 nuclear immunoreactivity at G2/mitosis transition. These data support the roles of CDK2 and cdc2 at G1/S and G2/mitosis transitions, respectively. (3) We were unable to demonstrate in individual cells a strict association between the nuclear appearance of cyclin A and G1/S transition, and an association of cyclin A and CDK2 with PCNA-stained DNA replication sites. On the other hand, the lengthening of G2 phase in the TSH/cyclic AMP-dependent thyroid cell cycle was associated with a stabilization of Tyr15 inhibitory phosphorylation of cdc2 and an especially high nuclear concentration of cyclin A and CDK2. We hypothesize that high nuclear accumulation of cyclin A and CDK2 during G2 phase could be causative in the cyclic AMP-dependent delay of mitosis onset. © 1996 Wiley-Liss, Inc.