Null MEFs Research Articles

Activation of the hypoxia-inducible factor 1 alpha is necessary for type 1 interferon and IFN stimulatory gene expression during gammaherpesvirus infection

Abstract Oxygen availability and the hypoxia-inducible factor 1 pathway (HIF1) influence the activity of cells involved in immune responses. Initially described as a master regulator of cellular adaptation to low oxygen concentration, activation of the hypoxia-inducible factor 1 alpha (HIF1α) is now recognized as an integral part of inflammatory cytokines production, phagocytic activity and bacterial killing by myeloid-derived cells. The goal of our studies is to determine the role of HIF1α during antiviral responses. We measure type 1 interferon gene expression in mouse embryonic fibroblasts lacking HIF1α activity (HIF1α Null MEFs) during early infection against the DNA virus, murine gammaherpesvirus 68 (MHV68). We recently reported that MHV68 activates HIF1atranscription activity and this is necessary for viral replication. In this study, analysis of mRNA levels by real-time qPCR, revealed that IFNβ1 decreased by 5-fold in HIF1α Null MEFs following 8 hours post infection (hpi), relative to wild-type, whereas levels of IFNα4, IFNα6 remained unchanged. Consistently, transcript levels of the IFN-stimulated gene OAS1β were reduced 7-fold relative to HIF1α WT MEFs. However, at 24hpi absence of HIF1α diminished expression of IFNα4, IFNα6, IFNβ1, OAS1β, IFIT18 and ISG20. In conclusion, these results suggest that HIF1α activity plays an important role during antiviral responses against dsDNA viruses.

Lamin A/C promotes DNA base excision repair.

The A-type lamins (lamin A/C), encoded by the LMNA gene, are important structural components of the nuclear lamina. LMNA mutations lead to degenerative disorders known as laminopathies, including the premature aging disease Hutchinson-Gilford progeria syndrome. In addition, altered lamin A/C expression is found in various cancers. Reports indicate that lamin A/C plays a role in DNA double strand break repair, but a role in DNA base excision repair (BER) has not been described. We provide evidence for reduced BER efficiency in lamin A/C-depleted cells (Lmna null MEFs and lamin A/C-knockdown U2OS). The mechanism involves impairment of the APE1 and POLβ BER activities, partly effectuated by associated reduction in poly-ADP-ribose chain formation. Also, Lmna null MEFs displayed reduced expression of several core BER enzymes (PARP1, LIG3and POLβ). Absence of Lmna led to accumulation of 8-oxoguanine (8-oxoG) lesions, and to an increased frequency of substitution mutations induced by chronic oxidative stress including GC>TA transversions (a fingerprint of 8-oxoG:A mismatches). Collectively, our results provide novel insights into the functional interplay between the nuclear lamina and cellular defenses against oxidative DNA damage, with implications for cancer and aging.

Loss of the Spinocerebellar Ataxia type 3 disease protein ATXN3 alters transcription of multiple signal transduction pathways.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disorder caused by a polyglutamine-encoding CAG repeat expansion in the ATXN3 gene which encodes the deubiquitinating enzyme, ATXN3. Several mechanisms have been proposed to explain the pathogenic role of mutant, polyQ-expanded ATXN3 in SCA3 including disease protein aggregation, impairment of ubiquitin-proteasomal degradation and transcriptional dysregulation. A better understanding of the normal functions of this protein may shed light on SCA3 disease pathogenesis. To assess the potential normal role of ATXN3 in regulating gene expression, we compared transcriptional profiles in WT versus Atxn3 null mouse embryonic fibroblasts. Differentially expressed genes in the absence of ATXN3 contribute to multiple signal transduction pathways, suggesting a status switch of signaling pathways including depressed Wnt and BMP4 pathways and elevated growth factor pathways such as Prolactin, TGF-β, and Ephrin pathways. The Eph receptor A3 (Efna3), a receptor protein-tyrosine kinase in the Ephrin pathway that is highly expressed in the nervous system, was the most differentially upregulated gene in Atxn3 null MEFs. This increased expression of Efna3 was recapitulated in Atxn3 knockout mouse brainstem, a selectively vulnerable brain region in SCA3. Overexpression of normal or expanded ATXN3 was sufficient to repress Efna3 expression, supporting a role for ATXN3 in regulating Ephrin signaling. We further show that, in the absence of ATXN3, Efna3 upregulation is associated with hyperacetylation of histones H3 and H4 at the Efna3 promoter, which in turn is induced by decreased levels of HDAC3 and NCoR in ATXN3 null cells. Together, these results reveal a normal role for ATXN3 in transcriptional regulation of multiple signaling pathways of potential relevance to disease processes in SCA3.

Loss of DNA polymerase β induces cellular senescence.

We aim to establish that accelerated aging and premature cellular senescence seen in individuals with Down syndrome is related to reduced DNA polymeraseβ. We report here that primary fibroblasts from Down syndrome individuals exhibit greater SA-β-gal staining (fourfold increase, P < 0.001), increased p16 transcript abundance (threefold increase, P < 0.01), and reduced HMGB1 nuclear localization (1.5-fold lower, P < 0.01). We also find that DNA polymerase β expression is significantly reduced in Down syndrome primary fibroblasts (53% decline, P < 0.01). To evaluate whether DNA polymerase β might be causative in senescence induction, we evaluated the impact of murine DNA polymerase β nullizygosity on senescence. We find that unexposed DNA polymerase β -null primary fibroblasts exhibit a robust increase in the number of senescent cells compared to wild-type (11-fold, P < 0.001), demonstrating that loss DNA polymerase β is sufficient to induce senescence. We also see an additional increase in response to hydroxyurea (threefold greater than WT-HU, P < 0.05). These data demonstrate that loss of DNA polymerase β is sufficient to induce senescence. Additionally, we report a significant induction in spontaneous DNA double strand breaks in DNA polymerase β null MEFs (fivefold increase from wild-type, P < 0.0001). Our findings strongly suggest that DNA polymerase β is causative in senescence induction, reasonably pointing to DNA polymerase β as a likely factor driving the premature senescence in Down syndrome. Environ. Mol. Mutagen. 59:603-612, 2018. © 2018 Wiley Periodicals, Inc.

Abstract 5437: MESP1 cooperates with loss of ARF in malignant transformation

Abstract Cancer is a highly complex disease involving multiple genetic and epigenetic abnormalities. Inactivation or expression of key regulators can be critical for the maintenance and survival of tumor. My research aims at characterizing one such novel gene, Mesoderm Posterior Basic Helix-Loop-Helix transcription factor-1 (MESP1), in lung cancer initiation and progression. MESP1 is transiently expressed during embryonic development and is the first sign of nascent mesoderm. It is a master regulator of cardiovascular development. Our lab has discovered through genome-wide identification of MESP1 targets that it is required in mesendoderm lineage development. However, whether MESP1 plays any role in adult physiology, diseases and cancer, has never been investigated. The Cancer Genome Atlas (TCGA) data show that MESP1 expression is upregulated in cancers of all mesendoderm-derived tissues. Bioinformatic gene expression analysis of clinical lung adenocarcinoma patient samples (396) showed that MESP1 was highly overexpressed in lung cancer. Similarly, RT-qPCR analysis of patient RNA samples from various stages of lung cancer showed that MESP1 expression was induced in early stages of lung cancer. It was found that MESP1 stable overexpression in ARF-null MEFs leads to oncogenic transformation of cells. This was evidenced by changes in various hallmarks of cancer such as increased cell proliferation, colony formation and anchorage-independent growth (about 6-fold increase in no. of colonies per field). In order to determine if the MESP1-induced oncogenic transformation was p53 dependent, we overexpressed MESP1 in p53-null MEFs. Interestingly, we saw no significant change in cell proliferation, colony formation and anchorage-independent growth in MESP1-overexpressing p53 null MEFs, suggesting that these effects are p53-independent. Upon performing global transcriptomic analyses in MESP1-overexpressing ARF-null MEFs, we found that cell adhesion and cell migration were among the top pathways that were significantly altered. Future studies will focus on in vivo studies to check for effect of MESP1-overexpression and/or knockdown on tumor formation and investigate the mechanism of MESP1-induced oncogenic transformation linked with increased cellular adhesion and migration. Lung cancer is the leading cause of cancer-related deaths in both men and women. More than 50% of cases are diagnosed only at an advanced stage, for which the survival rate of the patient is only 4%. Hence, knowledge of new driver oncogenes of lung cancer is imperative. These results suggest that MESP1 plays an important role in cancer initiation and progression. Ultimately, this research would pave the way for therapeutic intervention of key processes regulated by MESP1 involved in lung cancer. Citation Format: Neha Tandon, Benjamin Soibam, Robert Schwartz, Yu Liu. MESP1 cooperates with loss of ARF in malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5437.

Abstract 1362: Argonaute 2 controls RAS activation in mouse embryonic fibroblasts

Abstract The RAS gene family is among the most commonly mutated genes within cancer, and while much research has elucidated the major downstream pathways, including MAPK and PI3K, little progress has been made in successfully targeting mutant RAS in cancer. We recently identified an interaction between the N terminal domain of Argonaute 2 (AGO2), a core component of RNA-induced silencing complex (RISC), and the Switch II domain of KRAS. Furthermore, this interaction was found in all cell lines tested, expressing either wild-type (WT) or mutant KRAS. We found that stable knockdown of AGO2 in KRAS dependent cell lines lead to a decrease in KRAS protein expression with a subsequent decrease in cellular proliferation. Conversely, the overexpression of AGO2 in these cells lead to both an increase in KRAS expression and oncogenesis. In addition, this interaction inhibits the RNAi function of AGO2 by preventing microRNA unwinding in the presence of oncogenic KRAS compared to WT-KRAS. Despite a clear association between mutant KRAS and AGO2 mediating increased KRAS mediated oncogenesis, the precise function of this interaction remains unclear in normal physiology. In order to better assess the endogenous function of the KRAS-AGO2 interaction, we analyzed two mouse embryonic fibroblast cell lines (NIH 3T3 and MEF) with complete knockout of AGO2. Utilizing a Raf-1 RAS binding domain (RBD) pulldown method, we assessed activated WT-RAS levels in AGO2 null NIH 3T3 and MEF cells. We found that knockout of AGO2 lead to an increase in WT RAS-GTP activation compared to normal control cells. Immunoblot analysis also indicates that AGO2 null fibroblasts lead to increase in RAS downstream signaling through the MAPK/ERK and PI3K/AKT pathways. Furthermore, rescue of AGO2 knockout using full length mouse AGO2 decreased wild type RAS activation and its downstream signaling. Taken together, these observations suggest that the AGO2 interaction may suppress WT-KRAS activation, leading to maintenance of RAS-GDP levels. Using RNA-seq, proteome and microRNA analysis, we have begun to identify the pathways that may be involved in RAS activation in AGO2 null cells. Early analyses indicate that AGO2 controls WT-KRAS levels and activity through multiple mechanisms, laying the foundation for a better understanding of the RAS-AGO2 interaction in normal physiology. Citation Format: Ronald F. Siebenaler, Sunita Shankar, Vijaya L. Dommeti, Malay Mody, Arul Chinnaiyan. Argonaute 2 controls RAS activation in mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1362. doi:10.1158/1538-7445.AM2017-1362

Abstract A30: A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation

Abstract To identify the cellular components that participate in the regulation of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, we carried out a genome-wide RNAi depletion screen. We employed a rabbit monoclonal antibody specific for RPS6 [Ser235P/Ser236P] and high content microscopy to quantify rpS6 phosphorylation in the pancreatic ductal adenocarcinoma cancer cell line (PDAC) MiaPaCa-2. Applying a stringent selection, we retrieved over 600 genes wherein at least two RNAi gave significant reduction in S6 phosphorylation. This cohort is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. To identify mTORC1 regulators that did not require the TSC, we examined which of the positives identified in MiaPaCa-2 cells were also required for S6 phosphorylation in TSC1 null mouse embryo fibroblasts. Among the 541 such gene products examined in TSC1 null MEFs, RNAi pools directed against 79 were found to reduce S6 phosphorylation significantly in 2 or 3 replicates. Among these, GPCRs, other transmembrane proteins and several signaling proteins comprised 30%, elements concerned with DNA structure and transcription, RNA processing and translation together comprised 28%, and the remainder distributed among proteins concerned with membrane transport and traffic, protein traffic and modification, mitochondria, metabolism and the cytoskeleton. Surprisingly, the only elements among this cohort previously associated with the maintenance of mTORC1 activity are two of the numerous subunits of the vacuolar ATPase subunits and the CUL4 subunit DDB1. RNAis against another 84 TSC1null MEF targets were observed to reduce S6 phosphorylation in only one of three replicates, however an indication that this group also bears attention is the presence of RPS6KB1 itself, as well as Rac1 and MAP4K3, a protein kinase that supports amino acid signaling of mTORC1 to RPS6KB1. Our screen confirmed previously known and proposed regulators of S6 phosphorylation and mTORC1 in addition to previously unidentified mTORC1 related proteins. Our results suggest the potential operation of feedback pathways from mRNA translation, RNA processing and DNA repair in the regulation of mTORC1. Since many cancers exhibit mTOR hyperactivation, identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new drug targets and lead to the development of beneficial therapies for tumors sensitive to mTOR inhibition. In addition, we found 43 genes that matched the mTOR essentiality profile in 216 different cancer cell lines represented in the Achilles dataset. These genes may help delineate a more general cellular state characterized by co-dependency of mTOR dependency and the respective P-S6 positive genes therefore providing the rationale for selecting specific proteins for further analysis as candidate drug targets. Citation Format: Angela Papageorgiou, Pablo Tamayo, Jill Mesirov, Joseph Avruch, Joseph Rapley. A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A30.

Nuclear termination of STAT3 signaling through SIPAR (STAT3-Interacting Protein As a Repressor)-dependent recruitment of T cell tyrosine phosphatase TC-PTP

STAT3 is associated with embryo development and survival as well as proliferation and metastasis of tumor cells. In a previous study, we demonstrated that STAT3-Interacting Protein As a Repressor (SIPAR) enhances the dephosphorylation of STAT3 and negatively regulates its activity. However, it remains unclear how SIPAR inhibits phosphorylation of STAT3. Here we demonstrate that SIPAR directly interacts with T cell protein tyrosine phosphatase TC45 and enhances its association with STAT3. This interaction triggers an accelerated dephosphorylation process for STAT3. Furthermore, SIPAR inhibits the transcriptional activity of STAT3 in wild-type MEF cells but not in TC45 null MEF cells. These results suggest that SIPAR terminates the activation of STAT3 through a dephosphorylation process that is dependent upon interaction with TC45 in the nucleus.

SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination

Aberrant elevation of JARID1B and histone H3 lysine 4 trimethylation (H3K4me3) is frequently observed in many diseases including prostate cancer (PCa), yet the mechanisms on the regulation of JARID1B and H3K4me3 through epigenetic alterations still remain poorly understood. Here we report that Skp2 modulates JARID1B and H3K4me3 levels in vitro in cultured cells and in vivo in mouse models. We demonstrated that Skp2 inactivation decreased H3K4me3 levels, along with a reduction of cell growth, cell migration and malignant transformation of Pten/Trp53 double null MEFs, and further restrained prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, Skp2 decreased the K63-linked ubiquitination of JARID1B by E3 ubiquitin ligase TRAF6, thus decreasing JARID1B demethylase activity and in turn increasing H3K4me3. In agreement, Skp2 deficiency resulted in an increase of JARID1B ubiquitination and in turn a reduction of H3K4me3, and induced senescence through JARID1B accumulation in nucleoli of PCa cells and prostate tumors of mice. Furthermore, we showed that the elevations of Skp2 and H3K4me3 contributed to castration-resistant prostate cancer (CRPC) in mice, and were positively correlated in human PCa specimens. Taken together, our findings reveal a novel network of SKP2-JARID1B, and targeting SKP2 and JARID1B may be a potential strategy for PCa control.

Abstract 68: Tumor operational signaling pathways in an Nf1 mutant mouse model system

Abstract Purpose/Objectives: NF1 is recognized as a tumor suppressor, via its role as a Ras GTPase activating protein. However, the precise mechanisms critical to tumor development in NF1 patients remain unclear. NF1 heterozygosity confers susceptibility to tumorigenesis, promoted by genotoxins such as radiation. We mutagenized Nf1 heterozygous mice using radiation to generate a diverse panel of Nf1 null tumors. Using these novel reagents we interrogated signaling pathways essential for tumor development and propagation towards identification of potential targets for therapeutic approaches. Materials/Methodology: Cell lines were derived from solid tumors developing after focal irradiation of Nf1 heterozygous mice. Radiation-induced tumors included mammary carcinomas, squamous carcinomas, and sarcomas. We employed a high-throughput toxicity screen of 94 conventional and targeted FDA-approved chemotherapeutics directed across diverse core cellular signaling pathways. Drug sensitivity profiles were determined by quantifying cell numbers after a 72 hour drug exposure, and tumor cell lines, non-transformed wild-type and Nf1 null MEFs, and mutant Ras-transformed cell lines (added as controls) were compared. Validation of identified drug sensitivities was performed by enzyme activity assays, phosphoprotein analysis, and soft-agar colony formation. Results: Nf1 mutant tumors failed to demonstrate a common drug sensitivity signature, but instead revealed a heterogeneous pattern of differential sensitivities, with subgroups clustering independent of tumor histology. However, radiation-induced Nf1 null tumor lines exhibited increased sensitivity to inhibition of the Ras-Erk and PI3K-mTOR axis as compared to non-transformed controls. Enhanced sensitivity to agents targeting cell cycle and survival pathways was also observed selectively within the Nf1 null tumor cell lines. Conclusions: These studies indicate that Nf1 null tumors, even those of the same histology, do not intrinsically share a signature sensitivity profile to pharmacologic inhibition. This suggests that therapeutic targeting of signaling pathways in Nf1 mutant tumors is not likely to be defined strictly by Nf1 loss alone. Notably, these Nf1 null tumors evidence varied operational dependence upon PI3K-mTOR and Raf-MEK activity. Current efforts are directed towards further resolving essential mediators, with the goal of more precisely defining signaling pathways which drive progression of the malignant state. Citation Format: Steve Braunstein, Rana Mroue, Brian Huang, Sourav Bandyopadhyay, Jean Nakamura. Tumor operational signaling pathways in an Nf1 mutant mouse model system. [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 68. doi:10.1158/1538-7445.AM2014-68

T-cells null for the MED23 subunit of mediator express decreased levels of KLF2 and inefficiently populate the peripheral lymphoid organs.

MED23, a subunit of the Mediator coactivator complex, is important for the expression of a subset of MAPK/ERK pathway-responsive genes, the constituents of which vary between cell types for reasons that are not completely clear. MAPK/ERK pathway-dependent processes are essential for T-cell development and function, but whether MED23 has a role in this context is unknown. We generated Med23 conditional knockout mice and induced Med23 deletion in early T-cell development using the lineage specific Lck-Cre transgene. While the total cell number and distribution of cell populations in the thymuses of Med23flox/flox;Lck-Cre mice were essentially normal, MED23 null T-cells failed to efficiently populate the peripheral lymphoid organs. MED23 null thymocytes displayed decreased expression of the MAPK/ERK-responsive genes Egr1, Egr2, as well as of the membrane glycoprotein Cd52 (CAMPATH-1). MED23 null CD4 single-positive thymocytes also showed decreased expression of KLF2 (LKLF), a T-cell master regulatory transcription factor. Indeed, similarities between the phenotypes of mice lacking MED23 or KLF2 in T-cells suggest that KLF2 deficiency in MED23 null T-cells is one of their key defects. Mechanistic experiments using MED23 null MEFs further suggest that MED23 is required for full activity of the MAPK-responsive transcription factor MEF2, which has previously been shown to mediate Klf2 expression. In summary, our data indicate that MED23 has critical roles in enabling T-cells to populate the peripheral lymphoid organs, possibly by potentiating MEF2-dependent expression of the T-cell transcription factor KLF2.

Abstract C208: Targeting the actin cytoskeleton of neuroblastoma: Elucidating the mechanism by which a novel class of anti-cancer compounds induces tumor cell death.

Abstract The actin cytoskeleton is an ideal chemotherapeutic target due to its role in numerous biological processes essential for tumor cell growth and survival. Targeting actin however has been problematic due to unacceptable levels of toxicity associated with impacting actin containing structures essential for normal cell function. We have developed a novel class of compounds which target tropomyosin, the second core component of an actin microfilament. By targeting the cancer associated tropomyosin, Tm5NM1 we are able to discriminate between the actin filament populations in normal and transformed cells. We have demonstrated that our first in class anti-Tm compound, TR100, impacts actin filament integrity leading to tumor cell death in vitro and in vivo in neuroblastoma and melanoma models. In this study we investigate the molecular mechanisms by which disruption of the actin cytoskeleton with the next generation anti-Tm compounds leads to tumor cell death. Preliminary studies have indicated that these compounds initiate cell death via the intrinsic mitochondrial apoptotic pathway. SH-EP neuroblastoma cells treated with TR200 showed a dose dependent increase in both caspase activation and annexin V staining as measured by Western Blotting and FACS analysis respectively. Significant changes in mitochondrial membrane integrity were also observed using TMRE staining in SH-EP cells treated with 5 µM TR200 for 24h. Interestingly, mitochondrial permeabilization was independent of the presence of proapoptotic factors Bax and Bak as Bax/Bak null MEFs still displayed sensitivity to TR200. To delineate the signaling pathways leading to apoptosis, a kinexus phospho-antibody array was conducted using SH-EP cells treated with 10 µM DTR200 for 8 and 24h. DTR200 treatment for 8h reduced the phospho-activity of key intermediates of the MEK/ERK pathway, in particular a significant decrease in the phosphorylation of the RSK1/2 family of kinases. Preliminary data would suggest that the compounds at 24h are mediating their effect through the downregulation of the MEK/ERK and p38/JNK cell survival pathways. We are now validating these findings to elucidate which signaling pathways mediate the apoptotic response to the anti-Tm compounds. Understanding the molecular mechanisms involved in anti-Tm mediated cell death will allow us to determine how these compounds can be used in combination with existing therapies to lower the apoptotic threshold of a tumor cell and ultimately improve the outcome of patients with neuroblastoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C208. Citation Format: Melissa Desouza, Margaret Nguyen, Peter W. Gunning, Justine R. Stehn. Targeting the actin cytoskeleton of neuroblastoma: Elucidating the mechanism by which a novel class of anti-cancer compounds induces tumor cell death. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C208.

Abstract 1975: AICAR induces cell death in LKB1-deficient NSCLC.

Abstract AICAR is a well known AMPK activator, and AICAR-induced AMPK activation has been shown to inhibit cell proliferation or induce apoptosis through various mechanisms, such as the inactivation of metabolic enzyme involved ATP consumption, the inhibition of the of fatty acid synthesisPI3K/AKT pathway, the activation of cell cycle regulators, or the activation of the JNK pathway. LKB1 is an upstream AMPK kinase, and energetic stress-induced AMPK activation usually requires LKB1. Interestingly, AICAR has also been shown to induce caspase-3 cleavage in LKB1- null MEF and ovarian cancer cells, indicating AICAR can induce cell killing through an AMPK- independent mechanism. Because LKB1 is most frequently inactivated in non-small cell lung cancer, we evaluated the effect of AICAR in lung cancer cell lines. AICAR preferentially induced caspase-3 cleavage in LKB1- mutant NSCLC cell lines H157, A549, H460 and H1944, but not in LKB1- wild type cell lines H1299, H358, H1650 and H1792. Transient depletion of LKB1 by RNAi in H1299 and H1792 cells promoted AICAR-induced caaspase-3 cleavage in the absence of AMPK activation, supporting the notion that AICAR induces cell killing in LKB1-depleted cells through an AMPK- independent mechanism. In LKB1-null H460 cells, AICAR treatment led to a significant increase in S phase cell populations, and G1 cell cycle arrest by serum starvation is was sufficient to prevent AICAR-induced cell death. These data indicated that AICAR-induced cell killing in LKB1- deficient cells is cell cycle dependent. In summary, the ability of AICAR to preferentially induce cell death in LKB1- deficient NSCLC suggestsed that LKB1 deficiency may be a therapeutic target for the treatment of NSCLC. Citation Format: Fakeng Liu, Wei Zhou, Xiuju Liu, Shi-yong Sun, Fadlo Khuri, Yu-long He, Diansheng Zhong. AICAR induces cell death in LKB1-deficient NSCLC. [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 1975. doi:10.1158/1538-7445.AM2013-1975

&amp;lt;i&amp;gt;Gsta&amp;lt;/i&amp;gt;4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity

The alpha class glutathione s-transferase (GST) isozyme GSTA4-4 (EC2.5.1.18) exhibits high catalytic efficiency to-wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4-4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4-4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, activation of stress-related kinases in 4-HNE(Ac)3 treated Gsta4 null MEF were inversely correlated with the catalytic efficiency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4-4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE.

Stabilization of p21 (Cip1/WAF1) following Tip60-dependent acetylation is required for p21-mediated DNA damage response

The molecular mechanisms controlling post-translational modifications of p21 have been pursued assiduously in recent years. Here, utilizing mass-spectrometry analysis and site-specific acetyl-p21 antibody, two lysine residues of p21, located at amino-acid sites 161 and 163, were identified as Tip60-mediated acetylation targets for the first time. Detection of adriamycin-induced p21 acetylation, which disappeared after Tip60 depletion with concomitant destabilization of p21 and disruption of G1 arrest, suggested that Tip60-mediated p21 acetylation is necessary for DNA damage-induced cell-cycle regulation. The ability of 2KQ, a mimetic of acetylated p21, to induce cell-cycle arrest and senescence was significantly enhanced in p21 null MEFs compared with those of cells expressing wild-type p21. Together, these observations demonstrate that Tip60-mediated p21 acetylation is a novel and essential regulatory process required for p21-dependent DNA damage-induced cell-cycle arrest.

Akt1 as a putative regulator of Hox genes

In mammals, precise spatiotemporal expressions of Hox genes control the main body axis during embryogenesis. However, the mechanism by which Hox genes are regulated is poorly understood. To discover the putative regulator of Hox genes, in silico analyses were performed using GEO profiles, and Akt1 emerged as a candidate regulator of Hox genes in E13.5 MEFs. The results of the RT-PCR showed that 5′ Hoxc genes, including ncRNA were upregulated in Akt1 null MEF. Combined bisulfite restriction analysis (COBRA) and bisulfite sequencing showed that the CpG island of a 5′ Hoxc gene was hypomethylated in Akt1 null cells. These results indicate that Hox expression could be controlled by the function of Akt1 through epigenetic modification such as DNA methylation.

Lovastatin Induces Multiple Stress Pathways Including LKB1/AMPK Activation That Regulate Its Cytotoxic Effects in Squamous Cell Carcinoma Cells

BackgroundCellular stress responses trigger signaling cascades that inhibit proliferation and protein translation to help alleviate the stress or if the stress cannot be overcome induce apoptosis. In recent studies, we demonstrated the ability of lovastatin, an inhibitor of mevalonate synthesis, to induce the Integrated Stress Response as well as inhibiting epidermal growth factor receptor (EGFR) activation.Methodology/Principal FindingsIn this study, we evaluated the effects of lovastatin on the activity of the LKB1/AMPK pathway that is activated upon cellular energy shortage and can interact with the above pathways. In the squamous cell carcinoma (SCC) cell lines SCC9 and SCC25, lovastatin treatment (1–25 µM, 24 hrs) induced LKB1 and AMPK activation similar to metformin (1–10 mM, 24 hrs), a known inducer of this pathway. Lovastatin treatment impaired mitochondrial function and also decreased cellular ADP/ATP ratios, common triggers of LKB1/AMPK activation. The cytotoxic effects of lovastatin were attenuated in LKB1 null MEFs indicating a role for this pathway in regulating lovastatin-induced cytotoxicity. Of clinical relevance, lovastatin induces synergistic cytotoxicity in combination with the EGFR inhibitor gefitinib. In LKB1 deficient (A549, HeLa) and expressing (SCC9, SCC25) cell lines, metformin enhanced gefitinib cytotoxicity only in LKB1 expressing cell lines while both groups showed synergistic cytotoxic effects with lovastatin treatments. Furthermore, the combination of lovastatin with gefitinib induced a potent apoptotic response without significant induction of autophagy that is often induced during metabolic stress inhibiting cell death.Conclusion/SignificanceThus, targeting multiple metabolic stress pathways including the LKB1/AMPK pathway enhances lovastatin’s ability to synergize with gefitinib in SCC cells.

Platelet-Derived Growth Factor-BB Mediates Cell Migration through Induction of Activating Transcription Factor 4 and Tenascin-C

The acute response to vascular cell injury, which underpins vasculo-occlusive pathologies such as atherogenesis and restenosis after percutaneous coronary intervention, involves a complex series of molecular events that alter patterns of gene expression and favor a synthetic phenotype. One transcription factor that has been implicated in this process is the evolutionarily conserved mammalian stress response pathway regulator activating transcription factor 4 (ATF-4). Here, we show for the first time that both mRNA and protein levels of ATF-4 are induced in smooth muscle cells (SMCs) by the potent migratory factor PDGF-BB through PDGFR-β. PDGF-BB also stimulates the expression of tenascin-C (TN-C), an extracellular matrix glycoprotein that regulates the activity of focal adhesion complexes, facilitating the SMC migration that underlies negative vascular remodeling in response to injury. Overexpression of ATF-4 increased transcript levels of the four TN-C isoforms in rat vascular SMCs, and ATF-4 knockdown inhibited PDGF-BB-inducible TN-C expression in vitro and injury-inducible TN-C protein expression in the balloon-injured rat artery wall. Furthermore, we show that ATF-4 is required for PDGF-BB-inducible SMC migration in response to injury. PDGF-BB-induced migration was also compromised in ATF-4 null mEFs, and this effect was rescued by the addition of TN-C. Our findings thus demonstrate the role of ATF-4 in both injury- and PDGF-BB-inducible TN-C expression and cell migration.

The Gain of Function of p53 Mutant p53S in Promoting Tumorigenesis by Cross-talking with H-RasV12

The loss of wild type p53 tumor suppressive function and oncogenic gain-of-function of p53 mutants have been showing important implications in tumorigenesis. The p53N236S (p53N239S in human, p53S) mutation has been shown to lose wild type p53 function by yeast assay. However, its gain of function is still not clear. By gel shift assay, we showed that mutant p53S had lost its DNA binding ability to its target promoters. Further real-time PCR data confirmed that p53S had lost the function of regulating the transcription of p21 Cip1/Waf1, cyclin G, PUMA, and Bax in response to 10Gy irradiation. These data confirmed the loss of function of p53S in mammalian cells. By xenograft assay, we showed that the p53S per se was not oncogenic enough to form tumor, however, cooperating with H-RasV12, p53S could dramatically promote tumorigenesis in p53 null MEFs. Further study showed that co-expression of p53S and H-RasV12 could increase the expression level of H-RasV12 and partially eliminate the elevation of stress response proteins such as Chk2, γ-H2AX, Hsp70, Rb, p16Ink4a caused by either p53S or H-RasV12. These data suggested that p53S cross-talked with H-RasV12 and reduced the cellular stress response to oncogenic signals, which facilitated the cell growth and tumorigenesis. Together these data provided the molecular basis for the cooperation of p53S and H-RasV12 and revealed the gain of function of p53S in cross-talking with H-RasV12. This study revealed an important aspect of gain of function for p53 mutant, therefore might shed light on the clinical strategy in targeting p53 mutant.

Abstract 3: Interaction Between NFκB and NFAT Coordinates Cardiac Hypertrophy and Pathological Remodeling

Both NFAT and NFkB are Rel homology domain-containing family members of transcription factors whose independent activities are critically involved in regulating cardiac hypertrophy and failure. Here we identified a novel transcriptional regulatory mechanism whereby NFkB and NFAT directly interact and synergistically promote transcriptional activation of one another in cardiomyoctes. We showed that NFkB-p65 co-immunoprecipitates with NFAT isoforms in cardiomyocytes, and this interaction is mapped to the Rel homology domain (RHD) within p65. Intriguingly, overexpression of p65-RHD disrupts the association between endogenous p65 and NFATc1, leading to reduced NFAT luciferase activity. Overexpression of p65-RHD or IKKβ leads to significant nuclear translocation of NFATc1, and expression of a constitutively nuclear NFATc1-SA similarly facilitated p65 nuclear translocation, further suggesting a physical interaction between p65 and NFATc1. Indeed, we observed that combined overexpression of p65 and NFAT leads to synergistic activation of NFAT transcriptional activity in cardiomyocytes. Conversely, NFAT transcriptional activity in cardiomyocytes is significantly reduced by inhibition of NFkB with IkBαM or dominant negative IKKβ. Importantly, hypertrophic agonist-induced NFAT activity is also significantly reduced in NFkB-p65 null MEFs compared to wild-type MEFs, while adenoviral-mediated expression of p65 restored NFAT luciferase activity in p65 null MEFs. In vivo, cardiac-specific deletion of NFkB-p65 using a Cre-LoxP system caused a ∼ 50% reduction in NFAT activity in NFAT-luciferase reporter mice. Moreover, ablation of p65 in the mouse heart decreased the hypertrophic response following pressure overload stimulation, reduced the degree of pathological remodeling, and preserved contractile function. Taken together, our results suggest a direct interaction between NFAT and NFkB pathways that may serve as a novel signaling mechanism in cardiac hypertrophy and failure.