bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2055: Influence of intermittent hypoxia on differentiation in neuroblastoma
Authors: Bhaskara Vasantha Kumar , Mohanam Indra , Rao Jasti S. , Mohanam Sanjeeva
Published: July 2012

Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas derive from neural crest stem cells blocked at different stages of the sympathetic nervous system development. Neuroblastomas display a variety of histological features that range from undifferentiated tumors in patients with poor outcome to those containing differentiated cell types which show a better prognosis. Hypoxia has been linked to tumor progression and increased malignancy. Like the most common malignant solid tumors, neuroblastomas contain zones of chronic or acute hypoxia reflecting poor oxygenation. Transient hypoxia and subsequent reoxygenation are common phenomena in solid tumors that greatly influence the outcome of therapies. Differentiation is a key feature in pathologic classification and prognosis of neuroblastomas. The present study focuses on how intermittent hypoxia (IH) modulates the differentiation of neuroblastoma cells. IH-conditioned cells were derived by exposing tumor cells to 10 repeated cycles of hypoxia followed by reoxygenation. We found IH-induced changes in the expression of dedifferentiation markers and genes associated with neural crest-like phenotype in human neuroblastoma cells. The protein levels of HIF-1α, HIF-2α, tyrosine hydroxylase and c-kit were found upregulated in IH-conditioned human neuroblastoma cells as determined by western blotting. Similarly the expression of genes of neuronal crest phenotypic markers Oct-4, Notch-1and angiogenic molecule VEGF was found increased in IH-conditioned neuroblastoma cells. Expression of the cancer stem cell marker CD133 was also found increased in IH-conditioned neuroblastoma cells as evaluated by immunoflorescence analysis. Parallel studies were also performed in neuroblastoma cells exposed to hypoxia for 24h. It has been observed that IH-conditioned neuroblastoma cells exhibit increased expression of the proteins of dedifferentiation markers and genes associated with neural crest-like phenotype compared with neuroblastoma cells exposed to hypoxia for 24h. We cultured neuroblastoma cells, exposed the cells to defined doses of retinoic acid and studied neurite extension. A decrease in the levels of HIF-1α, and neuronal differentiation marker NF-M protein was observed in hypoxic neuroblastoma cells treated with retinoic acid. IH-conditioned neuroblastoma cells showed significantly less neurite extensions when compared with parental cells indicating that IH may suppress the differentiation of neuroblastoma cells. Taken together, our data suggest that IH inhibits differentiation and facilitates stem cell-like characteristics in human neuroblastoma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2055. doi:10.1158/1538-7445.AM2011-2055
Abstract 2058: The adaptation of small cell lung carcinoma (SCLC) cells to hypoxia is partially hypoxia inducible factor (HIF)-independent
Authors: Persson Matilda Munksgaard , Johansson Martin , Monsef Nastaran , Beckman Siv , Påhlman Sven , Pettersson Helen M.
Published: July 2012

Small cell lung carcinoma (SCLC) is an extremely aggressive tumor that is widely metastasized in early stages of the disease and the 5-year survival rate for these patients is only approximately 9-15%. Here we study the adaptive response of SCLC cells to lower oxygen levels, since tumor hypoxia (∼1% oxygen) is related to aggressive tumor behaviour and poor outcome in several tumor forms. Areas of hypoxia are a common characteristic in solid tumors and in response to hypoxia tumor cells induce expression of several genes involved in a variety of processes. This adaptive response is primarily regulated by the transcription factors hypoxia inducible factor (HIF)-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others lung adenocarcinoma, neuroblastoma and breast carcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia.

We investigated the viability of SCLC cells cultured at hypoxia and characterized the expression levels of HIF transcription factors in SCLC cells and specimens using quantitative real-time PCR, western blot and immunohistochemistry. Expression levels of well known hypoxia-driven genes were studied and to further characterize the hypoxic adaptive response, a gene expression profile of hypoxic SCLC cells was performed using microarray analyses. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison.

Our results show that SCLC cells have a high adaptive capacity to hypoxia, even though a modest induction of well known hypoxia-driven genes is observed. Interestingly, the HIF-1α and HIF-2α are differentially regulated in SCLC cells as compared to non-SCLC. In SCLC cells, no HIF-2α protein is detectable whereas HIF-1α protein levels are sustained and increased up to 96 hours. In addition, knock-down of HIF-1α at 0.1% oxygen are not affecting the viability of the SCLC cells and our gene expression profile of hypoxic SCLC cells has identified genes important for SCLC cells survival at hypoxia. Our data suggest that SCLC cells have an adaptive response to hypoxia that is partially HIF-independent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2011-2058
Abstract 2059: HO-3867-induced apoptosis in hypoxic ovarian cancer cells occurs via ubiquitin-dependent degradation of signal transducer and activator of transcription 3 (STAT3)
Authors: McCann Georgia , Cohn David , Hideg Kalman , Kuppusamy Periannan , Karuppaiyah Selvendiran
Published: July 2012

Objectives: Hypoxia plays a role in tumorigenesis and the development of resistance to radio- and chemotherapy. We have shown that human xenograft tumors in mice are severely hypoxic and that hypoxic exposure increases the phosphorylation of STAT3 at the Tyr705 residue in A2780 cell lines and is associated with chemotherapeutic resistance. HO-3867, a novel curcumin analogue designed with our institution, induces cell cycle arrest and apoptosis via inhibition of the STAT3 signaling pathway. The goal of this study was to understand the mechanistic pathways involved. Methods: The study was performed using A2780 and SKOV3 human ovarian cancer cell lines grown under normoxic (21% O2) and hypoxic (1% O2) conditions for 24 hours. Hypoxia-exposed cells were subsequently treated with 10 or 20 uM of HO-3867 for 2 and 24 hours. The expression levels of pSTAT3, associated proteins, and apoptotic markers were evaluated by Western blot and RT-PCR. Quantification of apoptosis was performed by staining with Annexin V. We also examined cell viability and involvement of ubiquitin-dependent degradation of pSTAT3. Results: Treatment with HO-3867 under hypoxic conditions resulted in decreased protein expression of pSTAT3 Tyr705 and Ser727. Rapid downregulation of STAT3 correlated with accelerated ubiquitin-dependent degradation of Tyr705-phosphorylated STAT3. Treatment was associated with decreased levels of STAT3-targeting proteins VEGF, Bcl-2, Bcl-xL, and cyclin D1. RT-PCR revealed decreased BCL-2 mRNA expression with treatment. There was no change in expression of proteins upstream of STAT3 including pJAK1, JAK1, pTYK2, TYK2, SOCS1 and SOCS2. An induction in apoptosis contributed to inhibition of cell proliferation as HO-3867-exposed cells exhibited elevated levels of cleaved caspase-3, caspase-7, and PARP as well as an increase in apoptotic cell count measured by flow cytometry. Conclusion: Under hypoxic growth conditions, HO-3867 demonstrates cytotoxicity via inhibition of STAT3. The use of HO-3867 may be a potential adjunct in the treatment of hypoxia-mediated chemotherapy-resistant ovarian cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2011-2059
Abstract 206: High mobility group A1(HMGA1) proteins localize at the mitochondria and inhibit the p53-mediated intrinsic-apoptotic pathway
Authors: Esposito Francesco , Tornincasa Mara , Borbone Eleonora , Pierantoni Giovanna Maria
Published: July 2012

The high mobility group A (HMGA) non-histone chromosomal proteins play key roles in chromatin architecture and orchestrate the assembly of nucleoprotein complexes involved in gene transcription, replication, and chromatin structure. HMGA overexpression and gene rearrangements are frequent events in human cancer, but the molecular basis of HMGA oncogenic activity has not been completely elucidated. Recently, we have defined a new physical and functional interaction between HMGA1 and p53. This interaction modulates the transcription of p53 target genes such as Mdm2, p21waf1, Bax, Bcl-2, and inhibits p53-mediated apoptosis. Here we report that HMGA1 does not act only at transcriptional level but it has also a direct, anti-apoptogenic role at the mitochondria. In fact, we found that in mouse embryonic fibroblast (MEF) and several tumour cell lines overexpressing HMGA1, this protein is present at cytoplasmic level other than in the nucleus. Moreover, we demonstrate that HMGA1 stably localizes at the mitochondria, in which it binds Bcl-2, displacing it from the binding to p53, thus counteracting the release of cytochrome C. In agreement with this observation, the HMGA1-overexpressing cells show a significant reduction of cytochrome C release, after lethal dose of Ultraviolet (UV) light, whereas HMGA1-depleted cells are more susceptibile to UV treatment.

These data define a new localization of HMGA1 at the mitochondria level that allows HMGA1 to inhibit by a new mechanism the p53-mediated intrinsic-apoptotic pathway.

This mechanism may also have a role in the resistance of malignant cells to chemo- and radio-therapy, in particularly for the tumor cells harboring wild-type form of p53.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2011-206
Abstract 2060: Comprehensive multiparametric assay of hypoxia and reactive oxygen species in live cells using different instrumentation platforms
Authors: Lebedeva Irina V. , Szczepanik Maciej , Pande Praveen , Patton Wayne F.
Published: July 2012

Hypoxia is a pathological condition in cells and tissue arising from oxygen deprivation. Cancer-associated hypoxia has been linked to drug treatment resistance, increased cell proliferation and metastatic potential, which overall contributes to poor patient prognosis. Many of the cellular responses to hypoxia are now known to be mediated by the production of reactive oxygen species at mitochondrial complex III. The importance of the maintenance of cellular oxygen supply requires further investigation into the role of ROS as hypoxia signaling molecules. Despite sustained interest in the association between hypoxia, ROS production and disease, methods for conveniently and reliably identifying and quantifying hypoxic cells as well as redox status in patients have remained elusive.

We designed and synthesized a series of non-fluorescent or weakly fluorescent aromatic compounds that become fluorescent once activated by hypoxic conditions. The probes were validated using a variety of in vitro cell-based hypoxia model systems involving exposure to hypoxia-inducing compounds (CoCl2, desferrioxamine or dimethyloxalylglycine) or by incubation in an anoxic environment (95% N2, 5% CO2). In the in vitro cell-based hypoxia model systems, all tested probes exhibited strong fluorescent cytoplasmic signal, readily detectable by different instrumental platforms. For the real-time profiling of both hypoxic and redox cellular status, these self-immolative hypoxia probes were combined with various ROS detection probes and stained samples were analyzed using conventional fluorescence microscopy or flow cytometry.

The described assay workflow facilitates rapid, reproducible, specific and relatively simple functional and quantitative detection of cellular oxygen status in a real-time format, and can readily be implemented on widely available instrumentation. The assay may be applicable to the screening of putative modulators of hypoxia or ROS production. Profiling of ROS is also possible when specific inhibitors of ROS are implied. The outlined fluorescence detection method offers an attractive approach for studying hypoxia and ROS pathways in a noninvasive manner, providing the ability for spatial and temporal resolution of cellular signaling events. The described multiparameter assay should find use in diverse applications relating to detection, quantification and localization of hypoxic cells in vivo and in vitro, as tools for the development of predictive assays or possibly as biosensors for early treatment monitoring and validation of potential endogenous hypoxia biomarkers and signaling molecules.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2060. doi:10.1158/1538-7445.AM2011-2060
Abstract 2067: Tumor cell survival under microenvironmental stress: Hypoxia and/or low glucose upregulate poline oxidase
Authors: Liu Wei , Sharma Anit , Phang James M.
Published: July 2012

Proline is a stress substrate which can be readily released from collagen in extracelluar matrix (ECM) initiated by matrix metalloproteinases accompanying tumor progression and invasion. Proline oxidase (POX) can metabolize proline to generate reactive oxygen species (ROS) for inducing apoptosis or autophagy; it also is a source of energy (ATP) under metabolic stress. However, the contribution of proline metabolism to tumorigenesis under concurrent hypoxia and low-glucose in the tumor microenvironment, has not been explored. In the present study, we showed that hypoxia and low glucose induced POX expression in HT29 colorectal cancer cells. The combination of low-glucose and hypoxia had additive effects on POX expression. Knockdown of HIF-1α, an important mediator of the hypoxic response, did not alter POX expression. Instead, inhibition of the activation of AMPK, the celluar energy sensor, by its antagonist (Compound C) blocked the upregulation of POX under conditions of either hypoxia or low glucose. Functional studies showed POX was consistently responsible for the maintenance of cell growth under hypoxia, low-glucose or with the combination of both. However, ATP and ROS assays showed that ATP production from POX was favored under low-glucose condition with or without hypoxia, whereas POX preferred ROS production with hypoxia. POX knockdown partially decreased hypoxia-induced prosurvival autophagy, which was also decreased by the antioxidant, N-acetylcysteine. Taken together, these results suggest that the induction of proline metabolism with hypoxia and/or low-glucose contributes an important mechanism by which tumor cells switch to a survival mode. These findings offer new insights into our understanding of energy metabolism in tumor cells beyond the well-recognized Warburg effect of aerobic glycolysis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2067. doi:10.1158/1538-7445.AM2011-2067
Abstract 2069: Regulation of nucleostemin by reactive oxygen species
Authors: Huang Min , Whang Patrick , Chodaparambil Jayanth V. , Pollyea Daniel A. , Kusler Brenda , Xu Liwen , Felsher Dean W. , Mitchell Beverly S.
Published: July 2012

Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that is a key regulator of cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells and embryonic stem cells. We have identified NS as a previously unreported target of redox regulation in transformed hematopoietic cells. NS oligomerization is present in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); exposure of these cells to reducing agents including N-acetylcysteine results in the dissociation of NS into monomers and dimers. On the other hand, exposure of other cell types that have relatively low levels of intrinsic ROS to low levels of H2O2 induces disulfide bond-mediated oligomerization of NS and, with prolonged exposure, immobilizes NS within the nucleolus, preventing its translocation to the nucleoplasm in response to nucleolar stress. Increasing levels of ROS also result in significantly increased NS protein stability. The redox-and oncogene-mediated regulation of NS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from four patients with chronic myelogenous leukemia in blast crisis in which the BCR-ABL oncogene is expressed. In both instances, increased intrinsic ROS levels were associated with markedly increased expression of NS protein and oligomerization that was reversed by exposure of cells to N-acetylcysteine. These results demonstrate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, increase the oligomerization of NS, enhance its stability and most likely impair its ability to regulate Mdm2-p53 interactions in the nucleus.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2069. doi:10.1158/1538-7445.AM2011-2069
Abstract 207: PTEN contributes to p53 activation during skin transformation
Authors: Robbins Delira F. , Gu Xin , Liu Jianfeng , Ponville Jacqulyne , Morris Kerri , Zhao Yunfeng
Published: July 2012

Our previous studies have shown that the tumor suppressor p53 is activated and translocates to the nucleus, as well as, mitochondria during skin carcinogenesis. However, how p53, a short-lived protein is stabilized, and whether it works with other tumor suppressors remains unclear. We hypothesize that PTEN contributes to TPA-induced p53 activation in the early stages of skin carcinogenesis. Following TPA treatment, PTEN expression in the whole cell lysate was reduced; however, nuclear PTEN expression was increased, and the p53-PTEN interaction was detected via immunoprecipitation in murine skin epidermal JB6 P+ cells, an effect accompanied by an increase in the proapoptotic protein and p53 transcriptional target, Bax. In cell transfection experiments, knocking down PTEN expression via siRNA was sufficient to suppress p53 transcriptional activity as evidenced by decreased Bax expression. Similar effects were seen when p53 transcriptional activity was blocked by pifithrin-α, a specific p53 inhibitor that blocks p53 nuclear translocation. In addition, TPA-induced PTEN expression was significantly reduced in cells that were pre-treated with pifithrin-α. Our results indicate that the tumor suppressor, PTEN, promotes p53 activation in the early stages of TPA-mediated skin tumorigenesis to regulate the expression and transcriptional activity of p53. Therefore modulators of the PTEN- p53 complex can be therapeutically useful in the treatment of skin carcinogenesis and early-stage tumor promotion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 207. doi:10.1158/1538-7445.AM2011-207
Abstract 2073: Curcumin analog 27 induced reactive oxygen species mediate loss of androgen receptor protein expression in human prostate cancer cells
Authors: Fajardo Alexandra M. , MacKenzie Debra A. , Deck Lorraine M. , Vander Jagt David L. , Thompson Todd A. , Bisoffi Marco
Published: July 2012

The androgen receptor (AR) is an established therapeutic target for prostate cancer. Therefore, agents that down-regulate AR expression may be effective for prostate cancer therapy. We show that the experimental therapeutic agent, curcumin analog 27 (ca27), induces the down-regulation of the AR in LNCaP and LAPC4 prostate cancer cell lines. Treatment with ca27 selectively inhibits AR mRNA expression, whereas ca27 did not alter mRNA levels for other steroid hormone receptors, such as the glucocorticoid receptor. In addition, ca27 decreases AR protein expression and increases AR protein ubiquitination within 3 hours. However, this rapid decrease in AR protein expression by ca27 is not mediated by proteasomal degradation. Interestingly, we observed an induction of reactive oxygen species (ROS) within 1 to 2 hours of ca27 treatment, which precedes AR degradation, leading to the hypothesis that ca27 mediates AR degradation through a cellular oxidative stress mechanism of action. In support of this hypothesis, the degradation of the AR following ca27 treatment is abrogated in the presence of the antioxidant, N-acetyl cysteine. Treatment with ca27 did not reduce cell viability by 24 hours suggesting that the increase in ROS and the inhibition of AR protein expression are independent of cell death. To further evaluate the cellular response to oxidative stress by ca27, we investigated the induction of a critical transcription factor activated as a cellular redox sensor, Nrf2. Treatment with ca27 lead to the increased transcriptional activation of Nrf2 as measured by an Nrf2-luciferase reporter assay. The ca27-induced Nrf2 activation was further supported by increased mRNA expression of the Nrf2-regulated genes NAD(P)H quinone oxidoreductase 1 and aldoketoreductase 1C1. This study begins to identify the mechanism of action of ca27-mediated AR protein degradation with the overall goal of gaining insight into targeting ROS-mediated AR down-regulation as a strategy for prostate cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2073. doi:10.1158/1538-7445.AM2011-2073
Abstract 2077: Interactions between sulfiredoxin, S100A4 and non-muscle myosin IIA regulate cancer cell adhesion and motility
Authors: Bowers Robert R. , Manevich Yefim , Townsend Danyelle M. , Tew Kenneth D.
Published: July 2012

Sulfiredoxin (Srx) is an antioxidant protein that reduces over-oxidized catalytic cysteine residues of peroxiredoxins I-IV and catalyzes reduction of some S-glutathionylated proteins. We previously showed that Srx plays a role in cell migration and interacts with non-muscle myosin IIA (NM IIA; Bowers et al AACR 2010). The purpose of the present study was to extend our knowledge of the effect of Srx on cell adhesion and migration. Immunoprecipitation of Srx from non-small lung cancer (A549 cells) revealed a number of Srx-interacting proteins including NM IIA and S100A4. NM IIA plays a central role in the control of cell adhesion and migration, and S100A4 is a key regulator of NM IIA activity that is linked to cancer metastases. Adhesion assays showed that A549 cells over-expressing Srx have reduced adhesion to fibronectin-coated dishes and decreased levels of Tyr397-phosphorylated focal adhesion kinase (p-FAK). Conversely, Srx knockdown by shRNA increased A549 cell adhesion to fibronectin and p-FAK levels. Srx over-expression in A549 cells increased actin stress fiber formation and altered focal adhesion formation. Depletion of Srx in A549 cells resulted in decreased actin stress fiber formation and also altered sites of cell:extracellular matrix adhesion. In silico molecular modeling of an Srx-S100A4 homodimer complex showed close proximity (∼13Å) of the single Cys99 of Srx to the Cys85 of S100A4. The calculated binding constant for the Srx-S100A4 interaction indicated reasonable affinity (KD∼ 62 nM). This affinity was not dependent upon disulfide bonding, since mutation of Srx residue Cys99 to Ser resulted in increased affinity (KD ∼22 nM). In vitro S-glutathionylation of Cys85 of S100A4 markedly increased its affinity to Srx (KD∼4 nM), but had no effect with the inactive Srx C99S mutant. Srx over-expression produced a doubling of the basal cellular level of free Ca2+ suggesting that the protein:protein interactions altered the capacity of S100A4 to bind Ca2+. Our data show that Srx may play important role in S100A4-mediated cancer cell cytoskeletal remodeling and motility and could potentially be a target for development of specific anticancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2011-2077
Abstract 2078: PERK-mediated autophagy promotes Myc-induced transformation and tumorigenesis
Authors: Hart Lori S. , Cunningham Tom , Datta Tatini , Liu Huayi , Koong Albert C. , Fuchs Serge , Diehl J. Alan , Ruggero Davide , Koumenis Constantinos
Published: July 2012

The Unfolded Protein Response (UPR) is a coordinated cellular homeostatic program initiated by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, with the ultimate fate of the cell depending on both the intensity and persistence of the stress. We have previously shown that tumor cell survival under hypoxic stress is dependent on the UPR kinase PERK, as the growth of PERK-null tumors is significantly inhibited in vivo. The transcription factor c-Myc, the target of chromosomal translocation and gene amplification during the development of many human cancers, upregulates genes involved in ribosome biogenesis and increases the rate of protein synthesis, therefore, we hypothesized the UPR is activated by c-Myc overexpression. Using in vitro and in vivo models of c-Myc expression, we demonstrate that c-Myc activates PERK-dependent phosphorylation of eIF2α and upregulates the downstream effectors, ATF4 and XBP1s. UPR activation contributes to cell survival during c-Myc induced transformation via the induction of autophagy, as measured by LC3 processing and p62 degradation. Inhibition of PERK dramatically reduces c-Myc induced transformation and tumor growth in nude mice. We are currently developing a lymphoma mouse model to test the strategy of PERK inhibition orthotopically. We have established a role for c-Myc in the cellular response to ER-stress and our results highlight a role for PERK inhibition in the treatment of malignancies characterized by c-Myc overexpression, such as lymphomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2078. doi:10.1158/1538-7445.AM2011-2078
Abstract 208: The function of p53 is intact in murine basal cell carcinomas in IR-treated Ptch1+/- mice
Authors: Wang Grace Y. , Wang Joy , Wang Lynn , Libove Eileen , Epstein Ervin H.
Published: July 2012

Basal cell carcinomas (BCC) are the most common cancer among people of European ancestry. Although mutational activation of Hedgehog (HH) signaling is pivotal for BCC carcinogenesis, p53 mutations also frequently are present in human and mouse BCCs. In humans, these p53 mutations are frequently of a “UV-signature” type, and human BCCs typically arise in sun-damaged skin containing epidermal clones of p53 mutant cells, implying that p53 is a crucial BCC tumor suppressor. In line with this notion, we found that deletion of p53 specifically in K14-expressing keratinocytes dramatically accelerates BCC carcinogenesis in ionizing radiation (IR)-treated Ptch1+/- mice. In IR-treated Ptch1+/- mice without p53 deletion, visible BCCs can also arise from but with longer tumor latency (9m vs. 5m) and a lower tumor incidence (10% vs. 100%) and mutiplicity. However it is not known whether loss of p53 signaling activity is required for the development of visible BCCs in p53+/+ mice. To address this question, we compared p53 signaling activity in IR-induced BCCs in Ptch1+/- p53+/+ mice vs. in BCCs in Ptch1+/- p53-/- mice after a second, DNA-damaging dose of IR. Using immunohistochemistry, we found the expected minimal IR-induced expression of p53, Puma, and Noxa as well as TUNEL-detected apoptosis in BCCs of p53-deleted mice. By contrast, IR induced robust expression of p53, Noxa, Puma, and apoptosis in BCCs from Ptch1+/- p53-intact mice. Without IR exposure, BCCs in p53+/+ mice had scattered p53+ cells and low level of expression of Puma and Noxa but no TUNEL+ cells. Our data thus suggest that IR-induced BCCs generated in Ptch1+/- p53+/+ mice retain their functional p53 signaling capacity and that these tumors are able to evade an intact DNA damage response.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 208. doi:10.1158/1538-7445.AM2011-208
Abstract 2081: The unfolded protein response relieves stress in tumor cells by stimulating angiogenesis
Authors: Wang Yugang , Ning Yu , Alam Goleeta , Visioli Fernanda , Nör Jacques E. , Polverini Peter J.
Published: July 2012

Abstract: During the avascular phase, tumor size is limited to 1-2 mm in diameter. When tumors exceed this size, the high stress imposed by limited glucose and oxygen in the microenvironment induces the unfolded protein response (UPR). Previous studies have shown that the UPR can promote survival, or activate apoptosis when there is prolonged or severe stress. Recent studies have suggested a role for the UPR in angiogenesis. However, the mechanism of UPR-regulated angiogenesis has not been extensively investigated. We hypothesize that tumor cells relieve stress caused by nutrient and oxygen deprivation by stimulating blood vessel growth via UPR-mediated induction of angiogenesis. To address this hypothesis, a human squamous cell carcinoma line, UM-SCC-81B, was subjected to glucose deprivation and mRNA array was used to evaluate expression of UPR proteins and angiogenesis mediators. This study revealed an increase in expression of several UPR proteins (Grp78, DDIT3, ATF3, ATF4, ATF6) ranging from 2 to 20-fold after 24 hours of glucose deprivation (0.1 mM glucose), and a 4.43, 3.06 and 2.59-fold increase in the expression of the pro-angiogenic VEGF, FGF2 and IL-6, respectively. Western blot, ELISA and q-PCR were used to verify these results. Western blot analysis showed a significant increase of Grp78 and DDIT3 protein expression. ELISA experiments revealed that after 24 hours of glucose deprivation, VEGF levels increased 3 folds as compared to the non-treated control, and that FGF2 and IL-6 were also upregulated (p < 0.05). q-PCR results further revealed that UPR markers (Grp78, DDIT3, ATF4) and angiogenesis mediators (VEGF, FGF2, IL-6) increased significantly (p < 0.05). Four other tumor cell lines (i.e. U87, glioma; MCF7, breast cancer; UM-SCC 11B and UM-SCC 17B, head and neck squamous carcinomas) were examined to determine the cell-type specificity of these responses. In all tumor cell lines examined, the upregulation of UPR proteins was associated with a significant increase in VEGF expression (p < 0.05). In conclusion, the UPR response is associated with the upregulation of critical pro-angiogenic factors in tumor cell lines. These data strongly suggest that targeting pathways involved in UPR regulation of angiogenesis is a promising strategy in combination with other conventional cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2081. doi:10.1158/1538-7445.AM2011-2081
Abstract 2084: Reactive oxygen species activate the IRE1-XBP1 pro-apoptotic pathway by recruiting TRAF2 to IRE1 and activating an IRE1-TRAF2-JNK pathway in human glioma cells
Authors: Kim Yeon Hyang , Koh Hyung-Kon
Published: July 2012

To investigate the function of the oxidative stress in the endoplasmic reticulum (ER) -related cell death of glioma, the human glioma cells were treated with hydrogen peroxide, TNF, and thapsigargin to raise intracellular levels of hydroxyl radical and ER stress, respectively. Hydrogen peroxide and thapsigargin increased cytosolic Ca2+, induced the activation of caspase 3, and caused apoptosis in a dose-dependent manner. In contrast, TNF affect neither cell viability nor the increase of cytosolic Ca2+. Both hydrogen peroxide and thapsigargin increased the expressions of ER-associated molecules including p-PERK, IRE1-α, p-eIF, ATF-1, p-CREB and Ero1-α in human glioma cells, whereas TNF did not increase the expressions of them. Hydrogen peroxide and thapsigargin increased IRE1-α nuclease activity generating transcriptionally active form of XBP-1 and induced IRE1-α-TRAF2 complex formation which initiates JNK activation and increase of expressions of p-JNK1 and p-JNK2. On the other hand, TNF-α did not increase p-JNK1and p-JNK 2 levels. IRE1-α induction coincided with those of p-JNK1, p-JNK and induced formation of IRE1-α-TRAF2 in hydrogen peroxide- and thapsigargin- treated cells. Therefore, we conclude that the increase of expression of IRE1-α is necessary for activating TRAF2-JNK pathway and causing oxidative stress- induced cell death of glioma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2011-2084
Abstract 209: Zinc is essential for the efficacy of p53 reactivating drugs
Authors: Azmi Asfar S. , Sukari Ammar , Philip Philip A. , Mohammad Ramzi M.
Published: July 2012

According to the International Zinc Nutrition Consultative Group, 20% of the world's population lack sufficient Zinc in their diet. Zinc is required by >200 different proteins including the most extensively studied Zinc-finger protein p53 that is considered guardian of genome. Its crucial role in p53 core domain folding has been proven, as disruption of this interaction greatly reduces or abrogates p53 DNA binding and transactivation of target genes. Therefore, alterations in Zinc homeostasis may impair p53 activity and also reduce the efficacy of wt-p53 re-activating drugs. Recently, we have shown that Zinc is necessary for proper wt-p53 re-activation and cancer cell apoptosis by small molecule inhibitors of HDM2 such as MI-219 (Oncogene 2010). As a logical extension to our these results, in this clinically translatable study we show that Zinc is crucial for the activity of mutant p53 reactivated by PRIMA and provide evidence for using combination therapies with PRIMA that include supplemental Zinc for the treatment of mut-p53 tumors. ZnCl2 (64 µM) enhanced PRIMA (0-100 µM) anti-tumor activity (induced growth inhibition and apoptosis and suppressed colony formation) in multiple pancreatic, colon and breast cancer cell lines. Chelation of Zinc by chelex 100 resin not only blocked the activity of PRIMA, but also suppressed reactivation of the mut-p53 and its downstream effector molecules 14-33σ, Bax and p21 as detected by western blot analysis. N,N,N'N′-tetrakis(−)[2-pyridylmethyl]-ethylenediamine (TPEN), a specific Zinc chelator, but not 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (Bapta-AM), a calcium chelator or desferroxamine, a iron chelator blocked PRIMA-induced apoptosis. Zinc chelation by TPEN suppressed p53 nuclear localization that is important for its transcriptional activity. Zinc did not affect PRIMA activity in p53 null cells, suggesting that this was indeed a p53 dependent phenomenon. Addition of Zinc suppressed the known p53 feedback MDM2 activation, which could be restored by TPEN. Before wt- or mut-p53 re-activating drugs such as MI-219 and PRIMA make their way into the clinic for cancer patients who usually have mild to moderate Zinc deficiency, we believe that the results of our findings will help in the design of novel combination strategies with Zinc for better survival outcome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 209. doi:10.1158/1538-7445.AM2011-209
Abstract 2094: Hsp70 inhibitors overcome tamoxifen resistance, in cell culture model
Authors: Koren John , Miyata Yoshinari , Kiray Janine , Guo Jianping , Gestwicki Jason , Cheng Jin Q. , Dickey Chad
Published: July 2012

A prevalent problem in the use of chemotherapies for the treatment of human cancers is the potential for the development of resistance. While the exact nature of the mechanisms involved in resistance development vary based on cancer type as well as the nature of the therapeutics; some oncogenic factors have been established to have roles in the desensitization to common treatments. Previous data from our lab demonstrated Hsp70 inhibitors can reduce Akt levels. a major proliferation and survival factor also linked to estrogen receptor therapeutic based resistance. We have, Uusing these Hsp70 ATPase-inhibiting compounds and ain a cell culture model of tamoxifen resistant breast cancer, we have identified compounds which can re-sensitize resistant cells. These compounds inhibit cytosolic and mitochondrial members of the 70-kDA heat-shock protein family. These compounds also reduce Akt levels, a major proliferation and survival factor also linked to estrogen receptor therapeutic based resistance, as well as Thus, in addition to reducing Akt levels and possibly other oncogenic chaperone clients, these drugs may also interrupt mitochondrial function leading to reduced viability in the presence of tamoxifen. With these mechanisms in mind, we now have data that Ttamoxifen resistant MCF7 cells regained sensitivity to tamoxifen after brief treatments with Hsp70-family inhibitors. The resistant cells were grown in media containing tamoxifen. This media was replaced with media containing an Hsp70 inhibitor compound for four hours. After the four hours, the Hsp70 inhibitor media was removed and tamoxifen-containing media was reapplied to the cells. Multiple viability and cytotoxicity assays confirm the drop in viability and increased cytotoxicity following these treatments. These studies demonstrate that Hsp70 family members are potentially valuable targets for new cancer therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2094. doi:10.1158/1538-7445.AM2011-2094
Abstract 2097: ARHI (DiRAS3): A novel component in the formation of the autophagy initiation complex
Authors: Lu Zhen , Liao Warren S. , Yang Maojie , Gao Fengqin , Bast Robert
Published: July 2012

The maternally imprinted Ras-related tumor suppressor gene Aplasia Ras homolog member I (ARHI; also known as DiRAS3) is downregulated in more than 60% of ovarian cancers. We reported that re-expression of ARHI in vitro at physiologic levels induces autophagy by blocking PI(3)K-Akt signaling and inhibiting mTOR activity, and participates directly in the autophagic vesicles, co-localizing with MAP-LC3-II in the vesicle membrane. Nucleation of autophagic vesicles is controlled by the activity and formation of multi-protein complexes that include Beclin-1 and several other positive and negative regulators. While binding of Beclin-1 to Vps34 and ATG14 forms proautophagic complexes to promote autophagy, binding of Bcl-2 to Beclin-1, however, disrupts the Beclin-1:Vps34 interaction and inhibits autophagy. The mechanisms that regulate the switch between different binding partners to Beclin-1, and therefore controls the level of autophagy is not clear. Here we report that ARHI physically interacts with endogenous Beclin-1 and Vps34 in ovarian cancer cells. Interaction with ARHI decreases the association between Beclin-1 and its negative regulator Bcl-2, by disrupting Beclin-1 homodimerization. An N-terminal deletion of ARHI that cannot induce autophagy fails to block Beclin-1 homodimerization and cannot disrupt Beclin-1:Bcl-2 interaction. Consistent with its role in promoting the formation of autophagy initiation complex, cells with knockdown of ARHI fail to respond to starvation-mediated dissociation of Beclin-1:Bcl-2 interaction and are unable to induce Beclin-1:Vps34 complex formation. Finally, ARHI knockdown decreases PI(3)KC3 activity and reduces starvation-mediated autophagy. Taken together, our results suggest that ARHI is a positive and necessary regulator of Beclin-1:Vps34 complex and that ARHI regulates starvation-mediated autophagy by promoting the formation of the autophagy initiation complexes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2097. doi:10.1158/1538-7445.AM2011-2097
Abstract 210: Metastasis-associated protein 1 is a transcriptional target of the tumor suppressor p53
Authors: Na Hyelin , Lee Min-Ho , Kim Kyu-Seo , Lee Jung Weon , Lee Mi-Ock
Published: July 2012

Although metastasis-associated protein 1 (MTA1) is known to play roles in angiogenesis and metastasis in various human cancers, the molecular mechanism of regulation of MTA1 gene expression remains unclear. Therefore, we searched potential regulators of MTA1 expression in this study. Treatment of 5-fluorouracil (5-FU), a traditional anti-cancer drug in HepG2, MCF7 and LNCaP cells, expressing wild-type p53, decreased the protein and mRNA expression of MTA1. However, the MTA1 expression levels were not altered in response to 5-FU treatment in Hep3B, MDA-MB-231 and PC3 cells which lack functional p53. The specific knockdown of p53 in HepG2 and MCF7 cells recovered the level of MTA1 after 5-FU treatment. This observation indicates that p53 mediates the repression of MTA1. Next, we virtually analyzed the promoter region of the human MTA1 gene and identified several potential consensus binding sequences of p53. Indeed, chromatin immunoprecipitation and pull-down assays showed that p53 directly bound to the MTA1 promoter region. These findings suggest that p53 is transcriptionally associated with regulation of MTA1 gene expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 210. doi:10.1158/1538-7445.AM2011-210
Abstract 2100: Role of pocket proteins in HPV-associated head and neck squamous cell carcinomas
Authors: Shin Myeong Kyun , Pitot Henry C. , Sage Julien , Lambert Paul F.
Published: July 2012

High-risk human papillomaviruses(HPVs) are associated with a subset of head and neck squamous cell carcinomas (HNSCC) as well as most of cervical cancers. HPV type 16 (HPV-16), which is found in the vast majority of HPV-positive HNSCC, encodes for three oncogenes: E5, E6, and E7. Previously, we learned that HPV-16 E7 is the dominant oncoprotein in head and neck cancer in mice. More recently, we learned that tissue specific inactivation of the retinoblastoma-susceptibility gene, pRb, a well known cellular target of HPV E7 and a member of the “pocket protein’ family, was not sufficient to account fully for the oncogenic properties of E7 in HNSCC. E7 is known to inactivate not just pRb, but also other related cellular proteins, specifically p107 and p130. This led to our current study in which we have observed that the combined loss of pRb and p130, or pRb and p107 leads to increased susceptibility to head and neck cancers in comparison to mice conditionally disrupted only for pRb, yet are not sufficient to account fully for E7's oncogenic potential in HPV-associated head and neck carcinogenesis. Additionally, combinatorial loss of pRb and p130, or pRb and p107 partially mediates E7-induced acute phenotypes in head and neck epithelia. Taken together, our results indicate that the combined inactivation of two pocket proteins are not sufficient to fully recapitulate E7 oncogenic properties in HNSCC. We conclude that inactivation of all three pocket proteins is necessary to account for E7's carcinogenic properties in HNSCC or that other targets of E7 contribute to its carcinogenic potential in HNSCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2100. doi:10.1158/1538-7445.AM2011-2100
Abstract 2106: The role of KSRP in non-small cell lung cancer
Authors: Wilson Lora A. , Winn Robert A.
Published: July 2012

Precedent has established the role of the Wnt/β-catenin pathway in oncogenesis. Several members of the Wnt family are known to be tumor promoting in non-small cell lung cancer, NSCLC. In this pathway β-catenin, a downstream component, accumulates and serves as a T-cell transcription /lymphoid enhancer factor. We previously identified the Wnt ligand in the lung, Wnt 7a, as a member of the non-canonical pathway/ β-catenin independent pathway, which inhibits transformed cell growth. Currently, we hypothesize that the regulatory protein, KSRP, holds a switch between the canonical and non-canonical pathway. K-homology splicing regulatory protein (KSRP) is a member of the family of AU rich element binding proteins. It possesses four K-homology motifs and causes decay of specific mRNA transcripts in different cell types. In mouse F9 teratocarcinoma cells KSRP negatively regulates β-catenin. The role of KSRP in the non-canonical Wnt pathway in the lung has previously been unknown. Using NSCLC cell lines and nontransformed lung cells we show a correlation between Wnt 7a expression and a positive regulation of KSRP. KSRP expression is elevated in nontransformed cells and alternatively, lower levels of KSRP expression in NSCLC. Over expression of KSRP in NSCLC cells decreases cell proliferation in MTS assay as well as reducing colony formation in clonogenic assays. The silencing of KSRP in nontransformed cells results in an increase in cell proliferation. The positive regulation of KSRP by Wnt 7a positively regulates this mRNA regulatory protein in the noncanonical Wnt pathway as an important factor in tumor suppression. The canonical pathway negatively regulates KSRP, which allows the stabilization of β-catenin mRNA thereby promoting progression of the tumor-promoting pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2011-2106