Impaired Cell Cycle Progression Research Articles

Transcriptional silencing of long noncoding RNA GNG12-AS1 uncouples its transcriptional and product-related functions.

Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription.

Enhanced efficacy of combined HDAC and PARP targeting in glioblastoma

Recent clinical trials have demonstrated that targeting chromatin remodeling factors is as a promising strategy for the treatment of glioblastoma (GBM). We and others have shown constitutive activation of DNA damage response (DDR) pathways in gliomas and suggested that targeting the DDR may improve the currently grim prognosis for patients. Based on our previous findings that inhibition of poly(ADP-ribose) polymerase (PARP) increases radio-sensitivity of the notoriously radio-resistant GBM cells, we hypothesized that epigenetic down-regulation of the DDR responses and induction of oxidative stress via HDAC inhibition would contribute to more efficient targeting of this deadly disease. Our data show that SAHA, an HDAC class I + II inhibitor, in combination with olaparib (PARP inhibitor): i) enhanced inhibition of GBM cell survival, ii) induced apoptosis, and iii) impaired cell cycle progression. These results provide a pre-clinical rationale for combined administration of SAHA and olaparib, which are already individually in clinical trials.

Role of the MSC-Derived Exosomal and Endogenous JAK2-SET/PP2A-Beta Catenin-Modulator Mir-300 in Leukemic Stem/Progenitor Proliferation and Survival in CML

MiR-300 is a microRNA predicted to target multiple components of the BCR-ABL1 / JAK2 / hnRNPA1 / SET / PP2A / β-catenin pathway, which is essential for survival/self-renewal of leukemic progenitors and quiescent TKI-resistant Ph+ hematopoietic stem cells (HSCs). Nanostring arrays analysis of bone marrow (BM) cells from healthy individuals (n=5) and CML patients (n=10) showed gradual inhibition of miR-300 expression (CML-CPmiR-300>CML-BCmiR-300).MiR-300 transduction in CMLCD34+ cells and BCR-ABL1+ cell lines decreased JAK2, β-catenin, hnRNPA1 and SET expression and increased PP2A activity. Targets were confirmed by miR-300 expression in BCR-ABL1+ cells expressing Flag-tagged miR-300-targets lacking or carrying a wild-type or mutated 3'UTR. Restored miR-300 expression in CMLCD34+ cells and/or BCR-ABL1+ cell lines impaired cell cycle progression, proliferation and clonogenic potential, markedly reduced LTC-ICs, and increased TKI sensitivity. Notably, miR-300 expression was inhibited by BCR-ABL1 in proliferating cells. Accordingly, imatinib restored miR-300 expression in CD34+ dividing progenitors and BCR-ABL1+ cell lines without altering miR-300 levels in quiescent (CFSEMAX) CMLCD34+ cells (n=3), consistent with the BCR-ABL1 kinase-dependent activation of the Jak2/SET/PP2A/β-catenin pathway in CML progenitors but not quiescent Ph+ HSCs. Indeed, ectopic SET expression counteracted the negative effects of mir-300 on cell proliferation and survival. Surprisingly, miR-300 levels were increased in CD34+CD38- compared to CD34+CD38+ CML cells, and >20-fold higher in CFSEMAX compared to dividing CMLCD34+ cells (n=4).To determine whether enhanced miR-300 expression in quiescent cells depends on cell autonomous events or is induced by the BM microenvironment, we exposed BCR-ABL+ cells to conditioned medium (CM) of HS-5 or hTERT mesenchymal stem cells (MSC). CM strongly decreased proliferation, induced imatinib but not FTY720 (PP2A activator) resistance, increased miR-300 levels, decreased BCR-ABL1 activity and Jak2 expression but not its activity, and did not alter b-catenin levels or PP2A activity. Interestingly, miR-300 was found in MSC-derived exosomes, and its expression increased in BCR-ABL1+ cells exposed to exosomes. Accordingly, proliferation of CML-BCCD34+ and LAMA-84 cells was strongly reduced upon exposure to MSC-derived exosomes. These effects were abolished when we used CM from MSCs transduced with a miR-300 antagomir.Altogether our results indicate that downregulation of miR-300 appears necessary for the activation of JAK2/SET/PP2A/b-catenin survival signals in CML progenitors. Conversely, increased miR-300 levels (endogenous and MSC-derived) seem to be required for HSC quiescence. DisclosuresDeininger:Novartis: Other: Consulting or Advisory Role, Research Funding; BMS: Other: Consulting & Advisory Role, Research Funding; Celgene: Research Funding; Genzyme: Research Funding; Gilead: Research Funding; ARIAD Pharmaceutical Inc.: Other: Consulting or Advisory Role; Incyte: Other: Consulting or Advisory Role; Pfizer: Other: Consulting or Advisory Role. Milojkovic:BMS: Honoraria; ARIAD Pharmaceuticals Inc.: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2′-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma.

Chromatin Remodeling Factor Brg1 Supports the Early Maintenance and Late Responsiveness of Nestin-Lineage Adult Neural Stem and Progenitor Cells.

Insights from embryonic development suggest chromatin remodeling is important in adult neural stem cells (aNSCs) maintenance and self-renewal, but this concept has not been fully explored in the adult brain. To assess the role of chromatin remodeling in adult neurogenesis, we inducibly deleted Brg1--the core subunit of SWI/SNF-like Brg1/Brm-associated factor chromatin remodeling complexes--in nestin-expressing aNSCs and their progeny in vivo and in culture. This resulted in abnormal adult neurogenesis in the hippocampus, which initially reduced hippocampal aNSCs and progenitor maintenance, and later reduced its responsiveness to physiological stimulation. Mechanistically, deletion of Brg1 appeared to impair cell cycle progression, which is partially due to elevated p53 pathway and p21 expression. Knockdown of p53 rescued the neurosphere growth defects caused by Brg1 deletion. Our results show that epigenetic chromatin remodeling (via a Brg1 and p53/p21-dependent process) determines the aNSCs and progenitor maintenance and responsiveness of neurogenesis.

Defective removal of ribonucleotides from DNA promotes systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease in which environmental exposures like virus infection and UV-irradiation trigger activation of the innate and adaptive immune system in genetically predisposed individuals. Heterozygous mutations of the 3' repair exonuclease 1 (TREX1) are associated with SLE. Biallelic mutations in TREX1 and the three subunits of ribonuclease H2 (RNASEH2A-C) cause Aicardi-Goutieres syndrome, an inflammatory encephalopathy with clinical overlap with SLE. We therefore investigated the role of RNase H2 in SLE pathogenesis. RNase H2 is responsible for the removal of misincorporated ribonucleotides from DNA and is indispensable for genome integrity. We demonstrated a genetic association for rare RNase H2 sequence variants with SLE. RNase H2-deficient fibroblasts of AGS and SLE patients accumulated ribonucleotides in genomic DNA resulting in chronic low-level DNA damage, constitutive p53 phosphorylation and senescence. Patient fibroblasts proliferated slower than fibroblasts from healthy individuals and showed impairment of cell cycle progression. In addition, patient fibroblasts exhibited constitutive up-regulation of interferon-stimulated genes and an enhanced type I interferon response to the nucleic acid poly(I:C) and UV-irradiation. UV-irradiation induced enhanced cyclobutane pyrimidine dimer formation in ribonucleotide-containing DNA. This suggests that innate immune activation may be caused by immune recognition of DNA metabolites of DNA damage repair and may also explain photosensitivity in SLE patients with RNase H2 mutation. In summary, our findings implicate RNase H2 in the pathogenesis of SLE, and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

Pharmacological inhibition of Bcl-xL sensitizes osteosarcoma to doxorubicin.

High-grade conventional osteosarcoma is the most common primary bone tumor. Prognosis for osteosarcoma patients is poor and resistance to chemotherapy is common. We performed an siRNA screen targeting members of the Bcl-2 family in human osteosarcoma cell lines to identify critical regulators of osteosarcoma cell survival. Silencing the anti-apoptotic family member Bcl-xL but also the pro-apoptotic member Bak using a SMARTpool of siRNAs as well as 4/4 individual siRNAs caused loss of viability. Loss of Bak impaired cell cycle progression and triggered autophagy. Instead, silencing Bcl-xL induced apoptotic cell death. Bcl-xL was expressed in clinical osteosarcoma samples but mRNA or protein levels did not significantly correlate with therapy response or survival. Nevertheless, pharmacological inhibition of a range of Bcl-2 family members showed that inhibitors targeting Bcl-xL synergistically enhanced the response to the chemotherapeutic agent, doxorubicin. Indeed, in osteosarcoma cells strongly expressing Bcl-xL, the Bcl-xL-selective BH3 mimetic, WEHI-539 potently enhanced apoptosis in the presence of low doses of doxorubicin. Our results identify Bcl-xL as a candidate drug target for sensitization to chemotherapy in patients with osteosarcoma.

Estrogen related receptor α (ERRα) a promising target for the therapy of adrenocortical carcinoma (ACC).

The pathogenesis of the adrenocortical cancer (ACC) involves integration of molecular signals and the interplay of different downstream pathways (i.e. IGFII/IGF1R, β-catenin, Wnt, ESR1). This tumor is characterized by limited therapeutic options and unsuccessful treatments. A useful strategy to develop an effective therapy for ACC is to identify a common downstream target of these multiple pathways. A good candidate could be the transcription factor estrogen-related receptor alpha (ERRα) because of its ability to regulate energy metabolism, mitochondrial biogenesis and signalings related to cancer progression. In this study we tested the effect of ERRα inverse agonist, XCT790, on the proliferation of H295R adrenocortical cancer cell line. Results from in vitro and in vivo experiments showed that XCT790 reduced H295R cell growth. The inhibitory effect was associated with impaired cell cycle progression which was not followed by any apoptotic event. Instead, incomplete autophagy and cell death by a necrotic processes, as a consequence of the cell energy failure, induced by pharmacological reduction of ERRα was evidenced. Our results indicate that therapeutic strategies targeting key factors such as ERRα that control the activity and signaling of bioenergetics processes in high-energy demanding tumors could represent an innovative/alternative therapy for the treatment of ACC.

DDX3Y, a Male-Specific Region of Y Chromosome Gene, May Modulate Neuronal Differentiation.

Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of the male-specific region of the Y chromosome (MSY) gene expression during neural development. In this study, we profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of NTERA-2 human embryonal carcinoma cell line (NT2) cells in three different developmental stages using qRT-PCR, Western blotting, and immunofluorescence. The expression level of 12 Y-linked genes significantly increased over neural differentiation, including RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. We showed that siRNA-mediated knockdown of DDX3Y, a DEAD box RNA helicase enzyme, in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 917 reproducibly identified proteins detected, 71 proteins were differentially expressed following DDX3Y siRNA treatment compared with mock treated cells. Functional grouping indicated that these proteins were involved in cell cycle, RNA splicing, and apoptosis, among other biological functions. Our results suggest that MSY genes may play an important role in neural differentiation and demonstrate that DDX3Y could play a multifunctional role in neural cell development, probably in a sexually dimorphic manner.

Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

Caffeine and Caffeic Acid Inhibit Growth and Modify Estrogen Receptor and Insulin-like Growth Factor I Receptor Levels in Human Breast Cancer.

Epidemiologic studies indicate that dietary factors, such as coffee, may influence breast cancer and modulate hormone receptor status. The purpose of this translational study was to investigate how coffee may affect breast cancer growth in relation to estrogen receptor-α (ER) status. The influence of coffee consumption on patient and tumor characteristics and disease-free survival was assessed in a population-based cohort of 1,090 patients with invasive primary breast cancer in Sweden. Cellular and molecular effects by the coffee constituents caffeine and caffeic acid were evaluated in ER(+) (MCF-7) and ER(-) (MDA-MB-231) breast cancer cells. Moderate (2-4 cups/day) to high (≥5 cups/day) coffee intake was associated with smaller invasive primary tumors (Ptrend = 0.013) and lower proportion of ER(+) tumors (Ptrend = 0.018), compared with patients with low consumption (≤1 cup/day). Moderate to high consumption was associated with lower risk for breast cancer events in tamoxifen-treated patients with ER(+) tumors (adjusted HR, 0.51; 95% confidence interval, 0.26-0.97). Caffeine and caffeic acid suppressed the growth of ER(+) (P ≤ 0.01) and ER(-) (P ≤ 0.03) cells. Caffeine significantly reduced ER and cyclin D1 abundance in ER(+) cells. Caffeine also reduced the insulin-like growth factor-I receptor (IGFIR) and pAkt levels in both ER(+) and ER(-) cells. Together, these effects resulted in impaired cell-cycle progression and enhanced cell death. The clinical and experimental findings demonstrate various anticancer properties of caffeine and caffeic acid against both ER(+) and ER(-) breast cancer that may sensitize tumor cells to tamoxifen and reduce breast cancer growth.

Highlights of This Issue

Breast cancer remains the leading cause of cancer-related death among women worldwide. Modifiable dietary components, such as coffee, may influence breast cancer and treatment responses. In a translational study, Rosendahl and colleagues uncovered associations between coffee consumption and tumor characteristics in breast cancer patients, as well as longer disease-free survival among tamoxifen-treated patients. Mechanistically, this was linked to altered estrogen- and insulin-like growth factor I receptor levels and downstream signaling, resulting in impaired cell-cycle progression and enhanced cell death. An improved understanding of how coffee can influence breast cancer and treatment response may yield more evidence-based lifestyle recommendations during treatment.It has long been advocated that normalization of peritumoral vessels through vascular endothelial growth factor (VEGF) inhibition may help to improve cytotoxic drug delivery to tumors. The best timing of giving VEGF inhibitor, however, remains unknown. In this clinical trial, Lu and colleagues tackled this issue by administrating bevacizumab one day before the combination of etoposide and cisplatin (BEEP regimen) to breast cancer patients whose metastatic brain tumors were refractory to whole brain radiotherapy. The BEEP regimen showed a central nervous system response rate of 77.1% by volumetric criteria and was associated with manageable toxicities.The pediatric tumor neuroblastoma presents with extraordinarily heterogeneous clinical courses, ranging from spontaneous regression to fatal disease progression. Accurate assessment of each individual patient's risk is therefore essential to appropriately tailor therapeutic intensities. To improve treatment stratification, Oberthuer and colleagues determined gene expression profiles of 709 primary neuroblastomas by microarrays and developed a gene expression-based classifier that predicts patient outcome with unprecedented precision. They developed a novel risk assessment system that combines the classifier with established markers, which may avoid over- and under-treatment of future neuroblastoma patients.Analyzing multiple tumor regions, Friemel and colleagues demonstrate intratumor heterogeneity in the majority of treatment-naïve hepatocellular carcinomas (HCC). Intratumor heterogeneity was not restricted to morphological features, immunohistochemical markers, or mutational aberrations of critical driver genes, CTNNB1 and TP53, but hierarchically linked between all three. The emergence of intratumor heterogeneity highlights the challenges associated with molecular classification of HCC and the development of targeted therapies because distinct HCC subpopulations may foster tumor adaptations resulting in therapy resistance or evasion.

Water-soluble isoindolo[2,1-a]quinoxalin-6-imines: In vitro antiproliferative activity and molecular mechanism(s) of action

Water-soluble isoindoloquinoxalin (IIQ) imines and the corresponding acetates were conveniently prepared from the key intermediates 2-(2′-aminophenyl)-2H-isoindole-1-carbonitriles obtained by a Strecker reaction between substituted 1,2-dicarbaldehydes and 1,2-phenylenediamines. Both series were screened by the National Cancer Institute (Bethesda, MD) and showed potent antiproliferative activity against a panel of 60 human tumor cell lines. Several of the novel compounds showed GI50 values at a nanomolar level on the majority of the tested cell lines. Among IIQ derivatives, methoxy substituents at positions 3 and 8 or/and 9 were especially effective in impairing cell cycle progression and inducing apoptosis in cancer cells. These effects were associated to IIQ-mediated impairment of tubulin polymerization at pharmacologically significant concentrations of tested compounds. In addition, impaired DNA topoisomerase I functions and perturbation in telomere architecture were observed in cells exposed to micromolar concentrations of IIQ derivatives. The above results suggest that IIQ derivatives exhibit multi-target cytotoxic activities.

Epigenetic dimension of oxygen radical injury in spermatogonial epithelial cells.

The present work reports a direct role of mitochondrial oxidative stress induced aberrant chromatin regulation, as a central phenomenon, to perturbed genomic integrity in the testicular milieu. Oxygen-radical injury following N-succinimidyl N-methylcarbamate treatment in mouse spermatogonial epithelial (GC-1 spg) cells induced functional derailment of mitochondrial machinery. Mitophagy resulted in marked inhibition of mitochondrial respiration and reduced mtDNA copy number. Impaired cell cycle progression along with altered H3K9me1, H4K20me3, H3, AcH3 and uH2A histone modifications were observed in the treated cells. Dense heterochromatin foci and aberrant expression of HP1α in nuclei of treated cells implied onset of senescence associated secretory phenotype mediated through nuclear accumulation of NF-κB. Neoplastic nature of daughter clones, emerged from senescent mother phenotypes was confirmed by cytogenetic instability, aberrant let-7a and let-7b miRNA expression and anchorage independent growth. Together, our results provide the first insights of redox-dependent epigenomic imbalance in spermatogonia, a previously unknown molecular paradigm.

Decreased expression of cell proliferation-related genes in clonally derived skin fibroblasts from children with Silver-Russell syndrome is independent of the degree of 11p15 ICR1 hypomethylation

BackgroundThe in vitro analysis of the hypomethylation of imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). To exclude mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and IGF2 and H19 expression were measured, and a microarray expression analysis was performed.ResultsSingle-cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from the patients and controls (Cnormo). IGF2 expression was below the detection limit of the quantitative real-time PCR (qRT-PCR) assay, whereas H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in SRShypo and Cnormo supernatants. Cell proliferation was diminished in SRShypo compared to Cnormo (p = 0.035). The microarray analysis revealed gene expression changes in SRS clones, predicting a decrease in cell proliferation and a delay in mitosis.ConclusionsThe analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal functional differences compared to normomethylated clones with respect to IGF2 and H19 expression. A difference compared to the clones from healthy individuals was present in the form of a lower proliferation rate, presumably due to impaired cell cycle progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0038-0) contains supplementary material, which is available to authorized users.

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.

Abstract 4497: Therapeutic benefit of targeting ERK in mutant KRAS pancreatic ductal adenocarcinoma

Abstract The limited success of anti-MEK and anti-RAF therapies with KRAS mutant cancers has been attributed, in part, to de novo or acquired resistance mechanisms that arise after prolonged treatment. A majority of resistance mechanisms involve kinome reprograming to reactivate ERK downstream of inhibitor activity. Therefore, we hypothesized that directly targeting ERK would be an effective method for overcoming these resistance mechanisms. To address this possibility, we evaluated the sensitivity of a panel of mutant KRAS pancreatic ductal adenocarcinoma (PDAC) cell lines to the highly selective SCH772984 ERK1/2 protein kinase inhibitor. First, we found that the anchorage-dependent growth of 6 of 12 cell lines were sensitive to SCH772984 treatment (IC50<2 uM). Some sensitive PDAC lines were resistant to the AZD6244 MEK1/2-selective inhibitor, indicating different mechanisms of action of these two inhibitors. Second, we identified phospho-AKT as a potential biomarker for PDAC SCH772984 sensitivity. Third, we assessed the molecular basis for anchorage-dependent growth inhibition. After 72 h treatment, flow cytometry analyses indicated impaired cell cycle progression through G1. Additionally, we found increased apoptosis as measured by formation of cleaved caspase-3. Together these observations suggest that sensitivity is a result of both impaired cell cycle progression and induction of apoptosis. Surprisingly prolonged SCH772984 treatment (2 weeks) resulted in senescence, as sensitive lines stained positive for senescence-associated beta-galactocidase. Furthermore, we found that senescent cells induced p16 and lost phospho-RB two common markers associated with induction of senescence. Onset of senescence was dependent on the loss of total MYC protein expression. Finally, we found that treatment with SCH772984 reduced tumorigenic growth in an in vivo xenograft mouse model. Our ongoing studies include determining the mechanisms of inhibitor resistance. In summary, our findings suggest that targeting ERK will be therapeutically beneficial for the treatment of KRAS mutant PDAC. Citation Format: Tikvah K. Hayes. Therapeutic benefit of targeting ERK in mutant KRAS pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4497. doi:10.1158/1538-7445.AM2014-4497

Abstract 4564: Inhibitory effects of the BTK inhibitor, ibrutinib, on Her2 amplified breast cancer growth, cell cycle progression and clonogenicity

Abstract Introduction: Ibrutinib is a Btk inhibitor developed for treatment of B cell malignancies. It binds covalently to cys-481 in Btk's kinase domain, which contributes to the drug's selectivity and enables once daily dosing. A homologous kinase domain cysteine is conserved in 9 other inhibitable kinases including those of the ErbB family. Ibrutinib thereby inhibits the in vitro growth of HER2 amplified breast cancer (BrCa) cells at pharmacologically relevant concentrations, and in vivo, in MB-453 xenografts, at dosages that model clinical exposure. To further characterize these effects we determined the effect of ibrutinib on growth, cell cycle progression, apoptosis and clonogenicity and stem cell-like subpopulations following treatment for short durations. Methods: BT-474 and SK-BR-3 BrCa cells were treated with ibrutinib at various times and concentrations, and culture continued after washing with fresh media. Cell growth was determined by alamar blue assay or direct cell counting. Cell cycle analysis was performed with PI staining and flow cytometry. Apoptosis was quantitated using flow cytometry with annexin-V/PI staining. Clonogenic assays were performed in 6-well plates with crystal violet staining. Putative “stem-like” cells were assessed by quantitating the aldehyde dehydrogenase 1 (ALDH1) population. Results: After drug washout, persistence of inhibition of ErbB kinase's phosphorylation and down-stream signaling was observed on a time and exposure dependent basis: while substantial washout-resistant inhibition occurred after 15 min treatment with 0.5 µM ibrutinib, over 1 hour was required for irreversibility following 0.1 µM treatment. Growth inhibition was sustained for 6 days following 1 hr exposure to 0.1 - 0.5 µM of ibrutinib. BrCa cells with short exposure were arrested in G1 with decreased S phase 24h later. ErbB kinases' activation and downstream signaling were also inhibited for up to 6 days. Although growth inhibition was largely cytostatic, 17% and 30% for BT-474 cells and SK-BR-3 cells, respectively, were apoptotic after 6 days at 0.5uM. Continuous treatment induced higher proportions of apoptotic cells. One hour washout or continuous treatment with ibrutinib decreased the putative “stem-like” subpopulation in BrCa cells as measured by Aldefluor assay. A short exposure of BrCa cells to ibrutinib also reduced their in vitro clonogenicity. Conclusions: Brief exposure to the Btk inhibitor, Ibrutinib, causes irreversible inhibition of ErbB kinases with consequent growth inhibition of HER2 amplified lines characterized by impaired cell cycle progression, evidence of apoptosis, and reductions of the Aldefluor population and clonogenicity. The unique combination of inhibitory properties of ibrutinib for both ErbB and TEC family kinases could prove to be advantageous in selected clinical settings. Citation Format: Jun Chen, Betty Y. Chang, Laurence Elias. Inhibitory effects of the BTK inhibitor, ibrutinib, on Her2 amplified breast cancer growth, cell cycle progression and clonogenicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2014-4564

Abstract 4393: miR-138 overexpression most effectively enhanced efficacy of apigenin to impair cell cycle progression and induce apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells in vivo

Abstract Malignant neuroblastoma, which mostly occurs in children, remains incurable with conventional chemotherapeutic agents. Innovative therapeutic strategies must be developed to control the growth of this childhood malignancy. Inhibition of cyclin D3 may induce cell cycle arrest and apoptosis in human malignant neuroblastoma. Also, inhibition of the catalytic component of human telomerase reverse transcriptase (hTERT) may induce apoptosis in many human cancers including malignant neuroblastoma. Decrease in expression of the tumor suppressor microRNA-138 (miR-138) is often associated with increases in expression of hTERT and cyclin D3. We hypothesized that miR-138 overexpression could be a better therapeutic strategy than hTERT knockdown to potentiate pro-apoptotic effect of apigenin (APG), a plant-derived flavonoid, in human malignant neuroblastoma. We found that transfection of miR-138 mimics was more powerful than transfection of hTERT shRNA plasmid in potentiating efficacy of APG for decreasing cell viability in human neuroblastoma SK-N-DZ and SK-N-BE2 cells. We then examined comparative effectiveness of different treatment strategies in reducing tumor growth and inducing apoptosis in SK-N-DZ and SK-N-BE2 xenografts in athymic nude mice. Combination of miR-138 overexpression and APG treatment showed better efficacy than combination of hTERT knockdown and APG treatment in reducing tumor growth and inducing cell death in both neuroblastoma xenograft models. According to the microRNA database (miRDB), miR-138 may target and inhibit expression of cyclin D3 so as to impair cell cylce progression. Combination of miR-138 overexpression and APG treatment caused the highest inhibition of expression of cyclin D3, CDK4, and CDK6, which were required for G1/S transition in cell cycle. Down regulation of cyclin D3 could inhibit phosphorylation of the tumor suppressor Rb and impair cell cycle progression. We delineated that apoptosis occurred with induction of molecular components of extrinsic and intrinsic pathways in the xenografts. Combination of miR-138 overexpression and APG treatment most effectively activated caspase-8 and cleaved Bid to tBid to induce extrinsic pathway of apoptosis and also raised Bax:Bcl-2 ratio to trigger intrinsic pathway of apoptosis with mitochondrial release of Smac into the cytosol to neutralize a set of endogenous inhibitor-of-apoptosis proteins such as Survivin so as to facilitate activation of caspase-3. Activation of both calpain and caspase-3 caused proteolysis of α-spectrin to generate calpain-specific 145 kD SBDP and caspase-3-specific 120 kD SBDP, respectively, in course of apoptosis in the xenografts. In conclusion, miR-138 overexpression was more powerful than hTERT knockdown in enhancing pro-apoptotic effect of APG for controlling growth of human malignant neuroblastomas in vivo. Citation Format: Mrinmay Chakrabarti, Walden Ai, Swapan K. Ray. miR-138 overexpression most effectively enhanced efficacy of apigenin to impair cell cycle progression and induce apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4393. doi:10.1158/1538-7445.AM2014-4393

Reduction of hRNase H2 activity in Aicardi-Goutières syndrome cells leads to replication stress and genome instability.

Aicardi–Goutières syndrome (AGS) is an inflammatory encephalopathy caused by defective nucleic acids metabolism. Over 50% of AGS mutations affect RNase H2 the only enzyme able to remove single ribonucleotidemonophosphates (rNMPs) embedded in DNA. Ribonucleotide triphosphates (rNTPs) are incorporated into genomic DNA with relatively high frequency during normal replication making DNA more susceptible to strand breakage and mutations. Here we demonstrate that human cells depleted of RNase H2 show impaired cell cycle progression associated with chronic activation of post-replication repair (PRR) and genome instability. We identify a similar phenotype in cells derived from AGS patients, which indeed accumulate rNMPs in genomic DNA and exhibit markers of constitutive PRR and checkpoint activation. Our data indicate that in human cells RNase H2 plays a crucial role in correcting rNMPs misincorporation, preventing DNA damage. Such protective function is compromised in AGS patients and may be linked to unscheduled immune responses. These findings may be relevant to shed further light on the mechanisms involved in AGS pathogenesis.