bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2054: The BH3-only protein, Noxa, is upregulated by estrogen and promotes cell cycle progression in estrogen receptor-positive breast cancer cells
Authors: Swetzig Wendy M. , Liu Wensheng , Medisetty Rajesh , Das Gokul M.
Published: August 2012

According to the American Cancer Society, breast cancer is the second leading cause of cancer-related deaths in American women, with more than 200,000 new cases diagnosed annually. In addition, the vast majority of breast cancers are initially hormone-dependent and 70% are estrogen receptor (ER)-positive (Colditz, J. Natl. Cancer Inst., 1998; Hankinson et al., Breast Cancer Res., 2004; and Harvey et al., J. Clin. Oncol., 1999), highlighting the importance of estrogen signaling pathways in breast oncogenesis. It has been well-established that estrogen/17β-estradiol/E2 stimulates cell growth and inhibits apoptosis in ER-positive breast cancer cells; however, recent reports have demonstrated that E2 paradoxically induces the expression of Noxa/PMAIP1, a BH3-only pro-apoptotic member of the Bcl-2 family. In the present study, we report on the mechanisms by which E2 upregulates Noxa expression and the associated cellular effects on apoptosis and cell cycle progression in ER-positive MCF7 human breast cancer cells. We demonstrate that c-Myc, ERα, and E2F1 are involved in the transcriptional upregulation of Noxa following E2 treatment. Specific knockdown of c-Myc and ERα protein expression using siRNA technology inhibited E2-mediated induction of Noxa expression. Furthermore, E2 also promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP) assays, suggesting that these two proteins are indeed mediators of Noxa upregulation following E2 treatment. Although p53 is an important regulator of Noxa transcription under conditions of cellular stress, our data demonstrate that E2-mediated upregulation of Noxa is a p53-independent process, as knocking down p53 with specific siRNA did not block E2-mediated induction of Noxa. Consistent with this result, E2 treatment failed to induce the recruitment of p53 to the NOXA promoter in ChIP assays. Interestingly, E2-mediated upregulation of Noxa was associated with cell cycle progression but not with apoptosis under normal, unstressed cellular conditions. Cell cycle analysis by flow cytometry showed that siRNA-mediated knockdown of Noxa caused cell cycle arrest in G0/G1-phase and significantly delayed the G1-to-S-phase transition following E2 treatment. Collectively, these data indicate that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells, suggesting a novel role for the BH3-only protein Noxa, outside of its traditional role as an apoptosis sensitizer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2054. doi:1538-7445.AM2012-2054
Abstract 2055: p21CIP1 mediates reciprocal switching between invasion and proliferation during metastasis
Authors: Hazan Rachel
Published: August 2012

Cell proliferation and invasion are critical for malignant progression, yet how these processes relate to each other and whether they regulate one another during metastasis is unknown. We show that invasiveness of breast cancer cells is associated with growth arrest due to p21CIP1 upregulation. Knockdown of p21CIP1 increases proliferation and suppresses invasion. Since p21CIP1 acts to inhibit Cyclin E during cell cycle progression, we demonstrated that a constitutively active form of Cyclin E had similar effects to p21CIP1 inhibition resulting in increased proliferation and suppressed invasion. We tested these findings in vivo in the Polyoma middle T mammary tumor model in which p21CIP1 was deleted. p21CIP1 knockout mice exhibited dramatic suppression of metastasis, independent of tumor growth. Metastasis suppression was associated with striking cytoskeletal reorganization leading to a shift from stress fibers to cortical actin, tightening of cell junctions, and cytokeratin upregulation. Thus, P21CIP1 regulates metastasis by mediating dynamic switching between invasion and proliferation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2055. doi:1538-7445.AM2012-2055
Abstract 2062: Peptidylprolyl cis/trans isomerase A1 (Pin1) is modified by lipid electrophiles generated during oxidative stress
Authors: Aluise Christopher D. , Marnett Lawrence J.
Published: August 2012

Oxidation of membrane phospholipids is associated with inflammation and cancer. Phospholipid peroxidation results in the production of reactive electrophiles that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we provide evidence that peptidyl-prolyl cis/trans isomerase A1 (Pin1), an enzyme that catalyzes the conversion of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE-modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry revealed that the most reactive amino acids are His-157 and Cys-113, both of which are active site residues. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and western blotting with anti-Pin1 antibody. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2062. doi:1538-7445.AM2012-2062
Abstract 2065: Loss of Nrf2 increases susceptibility to radiation induced bone loss
Authors: Rana Tapasi , Schultz MIchelle , Freeman MIchael , Biswas Swati
Published: August 2012

A fundamental clinical complication in the treatment of patients with metastatic breast cancer is severe bone loss. Patients with breast cancer routinely received both chemotherapy and radiotherapy, both are known to increase oxidative stress and bone loss. Underlying mechanism of cancer therapy induced bone loss is poorly understood. We have previouslyreported that treatment with radiation (RT) increases TGFβ1 levels in mice. TGFβ1 increases oxidative stress by induction of reactive oxygen species (ROS). TGFβ1 increases oxidative stress by induction of reactive oxygen species (ROS). TGFβ1/Smad3 signaling suppresses Nrf2 (Nuclear factor erythroid-derived 2), a master transcription factor that positively regulates induction of antioxidant gene expression resulting in elevated ROS levels. Our objective is to test the hypothesis whether loss of Nrf2 gene increases the radiation induced bone loss. We have used age matched wild type control and Nrf2-/- mice. Mice were irradiated (single dose, 20 Gy) on the hind legs and microCT analysis was done to measure the overall bone loss. A similar increase in bone loss was noted in mice following histological analysis of the long bones. Additionally, reduction in osteoblast colony forming units and osteoblast differentiation were also noted. We have seen a higher number of osteoclasts in Nrf2-/- mice compared to wild type control and following radiation, the trend remains same. We conclude that, loss of Nrf2 leads to incraesed susceptibility to the radiation induced bone damage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2065. doi:1538-7445.AM2012-2065
Abstract 2068: Opposite effects of arsenic trioxide on the Nrf2 pathway in oral squamous cell carcinoma in vitro and in vivo
Authors: Zhang Xinyan , Su Ying , Zhang Min
Published: August 2012

Nuclear factor erythroid derived like 2 (Nrf2) is a critical transcriptional factor in mediating cellular defense mechanisms against oxidative stress or electrophiles. Arsenic has been reported to induce malignant transformation of human cells through Nrf2-dependent signaling pathway. However, arsenic is also a promising cancer therapeutic drug for solid tumors, including oral squamous cell carcinoma (OSCC). It is still unclear how Nrf2 may mediate cellular response of OSCC cells when treated with arsenic. In order to fully understand the impact of Nrf2 signaling in human OSCC, we examined expression of Nrf2 and Nrf2-regulated genes in arsenic trioxide (ATO) treated OSCC cells in vitro and in ATO-treated OSCC xenografts. ATO exerted its anti-tumor effects on both cultured OSCC cells and OSCC xenografts by inhibiting cell growth, suppressing angiogenesis and inducing apoptosis. ATO activated a silent Nrf2 pathway in cultured OSCC cells as shown by induction of Nrf2 and Nrf2-regulated genes, NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), in a dose-dependent manner. On the contrary, Nrf2 pathway became active in OSCC xenograft tumors, and ATO treatment down-regulated expression of Nrf2 and Nrf2-regulated genes. Our study clearly demonstrated opposite effects of ATO on Nrf2 pathway in OSCC in vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2068. doi:1538-7445.AM2012-2068
Abstract 2071: Docosahexaenoic acid-induced apoptosis is directly linked to mitochondria-mediated reactive oxygen species production in human cervical cancer cells
Authors: Jing Kaipeng , Shin Soyeon , Kim Nayeong , Jeong Soyeon , Oh Hye-Rim , Cho Eun-Ah , Park Ji-Hoon , Seo Kang-Sik , Heo Jun-Young , Han Jeong-Su , Park Jong-Il , Kweon Gi-Ryang , Park Seung-Kiel , Yoon Wan-Hee , Hwang Byung-Doo , Lim Kyu
Published: August 2012

Reactive oxygen species (ROS) produced by docosahexaenoic acid (DHA) have an important function in cancer cell death. However, the exact mechanism of ROS production, after DHA stimulation, is not clearly understood. Here, we determined that elevated levels of ROS generated by mitochondrial respiration is directly associated with DHA-induced cervical cancer cell death. The levels of caspase 3 activity, TUNEL-positive staining cells and Sub-G1 portion were markedly increased in DHA-treated cancer cells, suggesting that apoptosis is responsible for the DHA-induced cervical cancer cell death. Furthermore, DHA was able to induce both mitochondrial complex I substrate- and complex II substrate-supported mitochondrial ROS production in isolated mitochondria from rodent liver. Meanwhile, a reduction in oxygen consumption rate and an increase in mitochondrial ROS production as measured by MitoSOX, were also observed in DHA-treated cancer cells, indicating that DHA can directly act on mitochondrial respiration and enhance ROS generation. The role of DHA-induced mitochondrial ROS production in apoptosis was further identified by the findings that DHA reduced the mitochondrial membrane potential, resulting in cardiolipin oxidation and cytochrome c release from mitochondria, and that N-acetylcysteine, an antioxidant almost completely blocked these processes as well as ROS production occurred in mitochondria and remarkably reversed the apoptotic cell death triggered by DHA. From the results presented here, we conclude that mitochondria actively participate in the DHA-induced apoptotic cell death by the generation of mitochondrial ROS. (This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (2011-0006232 and 2011-0003060)).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2071. doi:1538-7445.AM2012-2071
Abstract 2073: Parthenolide-mediated redox modification of Keap1 leads to differential effects on radiosensitivities of prostate cancer and normal cells
Authors: Xu Yong , Fang Fang , Clair Daret K. St. , Sun Yulan , Clair William H. St.
Published: August 2012

Elevated oxidative stress is more frequently observed in cancer cells than in normal cells. Thus, further exposure to exogenous reactive oxygen species (ROS) is expected to push cancer to cell death, whereas normal cells may maintain redox homeostasis through ROS mediated adaptive responses. We previously demonstrated that parthenolide selectively enhances radiosensitivity of prostate cancer cells through activation of a NADPH oxidase-dependent redox signaling pathway. Our present study identifies the status of the redox sensitive Kelch-like ECH-associated protein-1 (Keap1) as a central regulator responsible for the parthenolide-mediated differential redox modification. This results in both a radiosensitization effect in prostate cancer cells and a radioresistance effect in normal prostate epithelial cells. Mechanistically, parthenolide increases the reduced state of Keap1 in prostate cancer cells in a thioredoxin-dependent manner but causes oxidation of Keap1 in normal prostate epithelial cells. Selective knock-down of thioredoxin leads to Keap1 oxidation and subsequent activation of the NF-E2-related factor 2 (Nrf2). These results suggest that redox modification of Keap1 is a major mechanism for the parthenolide effect on radiation therapy in cancer and normal cells. Consistent with the differential effect of parthenolide in cancer and normal cells, the levels of superoxide, hydrogen peroxide and peroxynitrite were selectively increased in parthenolide treated prostate cancer cells but not in normal prostate epithelial cells, indicating the selective increase of ROS in cancer cells. Parthenolide reduces the levels of Nrf2 targets, antioxidant proteins including manganese superoxide dismutase and heme oxygenase in prostate cancer cells, but increases the levels of these proteins in normal cells. This suggests that an antioxidant response is critical for the protection of normal cells against ROS generated from parthenolide.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2073. doi:1538-7445.AM2012-2073
Abstract 2074: Cool-1-mediated inhibition of c-Cbl modulates multiple critical properties of glioblastomas, including the ability to generate tumors in vivo
Authors: Stevens Brett M. , Folts Christopher J. , Bardin Addie L. , Noble Mark
Published: August 2012

Glioblastomas (GBMs), the most malignant glial tumors, differ from normal glial progenitor cells in many ways, but little is known about the molecular circuitry underlying these differences. We have discovered a novel means by which GBMs become chemo-resistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1. The protein c-Cbl, an E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of multiple receptor tyrosine kinases critical for cell division and cell survival. In normal glial progenitor cells of the CNS, exposure to chemotherapy oxidizes cells, leading to sequential activation of Fyn and c-Cbl. However in GBM cells, exposure to BCNU or other chemical pro-oxidants does not lead to c-Cbl activation. Further studies have demonstrated that these agents do cause Fyn activation, suggesting that GBMs inhibit c-Cbl phosphorylation leading to decreased degradation of EGFR, accounting for increased EGFR signaling. We therefore set out to investigate the factors involved in the decreased c-Cbl activity specific to GBMs. In this work we discovered that oxidant-associated c-Cbl activation can be restored by suppressing expression of the protein Cool-1, found to be over-expressed in GBM. Our data further demonstrate that Cool-1 and c-Cbl formed complexes, that genetic reduction in Cool-1 levels restored normal c-Cbl-mediated degradation of RTKs (including EGFR) in response to exposure to pro-oxidants, and that restoration of normal c-Cbl activation has multiple effects on hallmarks of GBM. Cool-1 decreases lead to decreased migration and decreased numbers of cells in the cell cycle. Cool-1 inhibition leads to decreased number of cells that exhibit cancer stem cells characteristics including decreased antigen positivity and decreased sphere forming ability. In contrast, Cool-1/c-Cbl complexes were not found in normal brain or normal glial progenitors and Cool-1 knockdown did not reduce progenitor division. The translation of these findings into an in vivo intracranial xenograft model of GBM shows that Cool-1 is essential for tumorigenesis and decreasing Cool-1 levels leads to decreased tumor take, decreased tumor growth, and increased survival. In sum, the ability of Cool-1/c-Cbl interactions, to modulate a variety of tumor cell properties suggests that these interactions offer an attractive target for modulating GBM growth, particularly due to the absence of Cool-1/c-Cbl complexes in normal brain tissue and to the lack of effects of Cool-1 knockdown on division of normal glial progenitor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2074. doi:1538-7445.AM2012-2074
Abstract 2075: Overexpression of the peroxiredoxin family of antioxidant proteins in MCF7 breast cancer cells and investigation of possible regulatory mechanisms
Authors: Taparra Kekoa , Tehan Lauren , Rodriguez Braulio , Phelan Shelley A.
Published: August 2012

Peroxiredoxins are thiol-specific antioxidant proteins that protect cells from oxidative stress. There are six different peroxiredoxin proteins expressed in mammalian cells, and these proteins have been implicated in the regulation of cell signaling, proliferation and apoptosis. Many recent studies have found elevated peroxiredoxin levels in cancer, suggesting that cancer cells may upregulate peroxiredoxin expression as an adaptation to the cancerous state. We compared the expression of all six peroxiredoxins between MCF7 breast cancer cell line and the non-cancerous MCF-10A cell line. We found significant overexpression of five of the six peroxiredoxins (PrdxI-V) at both the mRNA and protein level. To explore possible mechanisms of peroxiredoxin upregulation, we first compared the lines for levels of reactive oxygen species (ROS) and the activity of particular kinases implicated in cancer biology. We found that MCF-7 cells express markedly higher levels of ROS, and increased activity of ERK 1/2 and JNK 1/2, as compared to MCF-10A cells. We hypothesized that elevated ROS and/or enhanced activity of these signal transduction pathways may mediate peroxiredoxin induction in these cells. MCF-7 cells were cultured under conditions of serum deprivation, ROS reduction, or kinase inhibition to examine the effects on peroxiredoxin expression. After 48 hours of serum deprivation, the expression of Prdx2 was reduced. Using NAC treatment, we inhibited ROS accumulation in MCF-7 cells and found a decrease in expression of both Prdx2 and Prdx3. Inhibition of ERK 1/2 activity by the inhibitor PD 98,059 resulted in little to no change in peroxiredoxin levels. Finally, treatment of MCF-7 cells with hydrogen peroxide led to a transient increase in Prdx1 at 4 hours, which returned to basal levels by 24 hours. Together, these data reveal overexpression of the peroxiredoxin family in MCF-7 cancer cells, and suggest that oxidative stress and redox-sensitive kinases may play a role in mediating the upregulation of particular peroxiredoxins. Studies examining the role of JNK and p38 in peroxiredoxin induction, as well as cell line differences in multimeric Prdx complexes, are currently underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2075. doi:1538-7445.AM2012-2075
Abstract 2076: Oxidative stress induced by sodium ascorbate:menadione sodium-bisulfate combination results in lysosomal and mitochondrial changes that lead to prostate cancer cell death
Authors: McGuire Karen , Neal Deborah , Fraizer Gail C. , Gilloteaux Jacques , Taper Henryk , Jamison James
Published: August 2012

Prostate cancer is a leading cause of death in American males and therapy targeting hormone refractory prostate cancer is urgently needed. Treatment of prostate cancer cell lines (LNCaP, LNCaP-C42, DU-145 and PC3) with either sodium ascorbate or menadione sodium-bisulfate alone induced cell killing (as measured by the MTT cell proliferation assay) only at high concentrations, while the combination (apatone) potentiated cytotoxicity 4- to 20-fold with exposure times as short as 1hr. Apatone treatment induces the reactivation of DNase I and II resulting in autoschizis, a caspase independent form of cell death. Cell cycle arrest (G1/S and G2/M blocks), increased hydrogen peroxide and lipid peroxidation levels, decreased cellular thiol levels, and increased intracellular Ca2+ levels were observed following apatone treatment. To further understand the mechanism(s) of action of this cytotoxic drug, the effects of apatone treatment on the lysosomal and mitochondrial membranes in DU-145 prostate cancer cells were measured by live cell fluorescence microscopy. Examination of lysosomal membranes using the metachromatic fluorochrome, acridine orange, revealed rapid permeabilization of the lysosomal membrane. The effect of apatone on mitochondrial membrane potential was also examined, using JC-1 staining, and revealed de-polarization of the mitochondria membrane, which occurred on a delayed time scale when compared to lysosomal activity. The in vivo anti-tumoral activity of apatone was evaluated in nude mice inoculated subcutaneously with DU-145 cells. Oral apatone significantly reduced the growth rate of the solid tumors (p<0.05) without inducing significant bone marrow toxicity, changes in organ weight or pathologies associated with these organs. Overall, these results support an apatone induced cell death mechanism involving a non-apoptotic cell death pathway initiated by oxidative stress in a pro-oxidant tumor environment. This stress ultimately leads to damaged lysosomes, mitochondria and other cellular organelles. This novel mechanism of action has been demonstrated to be highly effective both in vitro and in vivo, indicating that the sodium ascorbate:menadione sodium-bisulfate combination (apatone) can provide another urgently needed therapy for the treatment of hormone refractory and possibly hormone dependent prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2076. doi:1538-7445.AM2012-2076
Abstract 2080: The role of ErbB2 in regulating survival in matrix-detached inflammatory breast cancer cells
Authors: Buchheit Cassandra L. , Angarola Brittany , Steiner Allison , Schafer Zachary
Published: August 2012

Inflammatory breast cancer (IBC) is a rare and highly invasive type of breast cancer, and patients diagnosed with IBC often face a very poor prognosis. IBC is characterized by the lack of primary tumor formation and the rapid accumulation of cancerous epithelial cells in the dermal lymphatic vessels. Given that normal epithelial cells require attachment to the extracellular matrix (ECM) for survival, a comprehensive examination of the molecular mechanisms underlying IBC cell survival in the lymphatic vessels is of paramount importance to our understanding of IBC pathogenesis. Here we demonstrate that in contrast to normal mammary epithelial cells, IBC cells evade ECM-detachment-induced apoptosis (anoikis) and maintain ATP levels following ECM detachment. Exogenous ErbB2 overexpression leads to increased ATP production and soft agar growth in IBC cells that normally lack ErbB2 amplification. Additionally, ErbB2 knockdown in KPL-4 cells, an IBC cell line characterized by high levels of ErbB2, resulted in diminished ATP levels, decreased colony growth, and increased caspase activation following ECM detachment. Further studies reveal that the protection from anoikis in IBC cells is dependent on activation of the ERK-MAPK pathway, but does not depend on modulation of the levels of the pro-apoptotic factor Bim. These results demonstrate that ErbB2 is necessary and sufficient for IBC cell survival in the absence of ECM attachment. Understanding the molecular mechanism utilized by IBC cells to survive in matrix-detached conditions could lead to more specific and effective therapeutics to prevent IBC metastasis from occurring and to treat patients in which metastasis has already occurred.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2080. doi:1538-7445.AM2012-2080
Abstract 2081: Mechanisms of the acquisition of anoikis resistance in breast cancer: Lessons from small molecule inhibitors
Authors: West Winston , Park Margaret A.
Published: August 2012

The Cox-2 inhibitor Celecoxib has been reported to possess chemopreventive and chemotherapeutic properties, specifically in hormonally-regulated tumors. OSU-03012 is a novel celecoxib derivative, with no COX-2 inhibitory activity, but an order of magnitude greater cell killing ability. We here show that this novel compound has a greater cytotoxicity against a model of anoikis-resistant (AnR) breast cancer. In parallel, anoikis resistance reduces AnR cell sensitivity to the ErbB1/2 dual inhibitor Lapatinib (GW572016, Tykerb, GSK). The two compounds synergize in parental breast cancer cells. Cell killing is achieved partially via induction of ER stress, as measured by eIF2-alpha phosphorylation. We have found that lapatinib resistance may be induced by the aberrant splicing of BCL-X, and that the sensitivity to OSU-03012 is likely due to PTEN dysregulation. In conclusion, these data identify a molecule that may be more efficacious against metastatic tumors, and partially defined its mechanism of action.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2081. doi:1538-7445.AM2012-2081
Abstract 2091: Ultrasensitive detection of mutations in the androgen receptor gene by ICE COLD-PCR
Authors: Shi Yanggu , Cubrich Courtney , Veys Stephanie , Richardson Katherine , Legendre Benjamin , Lin Rui , Wu Grant , Eastlake Phil
Published: August 2012

The Androgen Receptor plays an important role in prostate cancer, androgen-dependent (AD) and androgen-independent (AI) disease progression. Anti-androgen therapies are the hallmark of prostate cancer treatment; however spontaneous AR mutations are often detected in hormone-refractory, androgen-ablated and metastatic tumors. ICE COLD-PCR (Improved & Complete Enrichment CO-amplification at Lower Denaturation temperature) is a technology that preferentially enriches mutant DNA sequences in an excess of wild-type DNA through selective amplification of the mutant DNA population using an oligonucleotide complementary to wild-type sequence (RS-oligo). This RS-oligo prevents PCR amplification of wild-type sequences while allowing amplification of DNA containing any mutation covered in the RS-oligo region. We have developed an ICE COLD-PCR assay for the enrichment of mutations in the Androgen Receptor including the AR H875R, H875Y and T878A mutations. The mutant-enriched DNA was then analyzed by standard Sanger DNA sequencing. The analytical sensitivity for the ICE COLD-PCR assay was 1 in 10,000 (0.01%) for all the mutations, compared to 20% limit of detection by standard Sanger sequencing. The ICE COLD-PCR assay developed here provides a means to detect low level mutations in the Androgen Receptor and thus will provide a valuable methodology for the early diagnosis, treatment guidance, outcome prediction and relapse monitoring of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2091. doi:1538-7445.AM2012-2091
Abstract 2094: Comparing exome with whole-genome next-generation sequencing in detecting somatic mutations
Authors: Yue Yong Gang , Ting Jason , Ma Xiwen , Barber Thomas D.
Published: August 2012

Exome sequencing has been used as an efficient and cost-effective method of identifying somatic mutations in tumors. In order to evaluate the effectiveness of the current exome sequencing technology, we have conducted a comparative study to evaluate exome and whole genome sequencing (WGS) in two pairs of hepatocelluar carcinoma (HCC) tumor and match normal controls. The exome sequences were captured via Agilent SureSelect Target Enrichment System (38 MB). Both exome and WGS sequencing data were generated by the Illumina Hiseq2000 platform. The NGS results showed identical profiles of chromosomal abnormalities, including ploidy changes and loss of heterozygosity, when compared to Affymetrix SNP 6.0 microarray genotyping. The total number of variants detected in transcribed region (CCDS) in each of the four samples was comparable between exome and WGS data sets, and the genotypes concordance rate was high. The somatic mutations detected in each tumor/normal pairs different slightly between the exome and WGS platforms. We manually inspected each of the discordant mutations, and found that the most of the somatic mutations missed by exome sequencing are predictable based on gaps in SureSelect coverage. On the other hand, we have also identified somatic mutations in exome that were not detected with the WGS approach. Follow up analysis indicated that those cases were caused by no or low coverage in either tumor of normal in each pair. These cases will likely be resolved by increasing depth of coverage (beyond 30X), and as costs per read decline, it is likely WGS will become the method of choice.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2094. doi:1538-7445.AM2012-2094
Abstract 2098: Reducing sequence artefacts from FFPE DNA for cancer diagnostics
Authors: Do Hongdo , Dobrovic Alexander
Published: August 2012

Non-reproducible sequence artefacts have been frequently detected in DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Studies of ancient DNA reveal that deamination of cytosine to uracil is a major cause of DNA damage. We hypothesised that similar lesions may occur in FFPE DNA and examined the role of uracil lesions on generation of transitional G:C to A:T artefacts in FFPE DNA by treatment with uracil DNA glycosylase (UDG). High resolution melting (HRM)-based assays were used for rapid and cost-effective screening of artefacts. The artefact rate was assessed by limited copy number-HRM (LCN-HRM) in FFPE DNA samples in which multiple G:C to A:T artefacts were found. The effect of UDG treatment on detection of true sequence changes was examined by testing mutational hotspots at the EGFR, KRAS and AKT1 genes. A high HRM positive rate as judged by the formation of heteroduplexes was found in FFPE DNA by LCN-HRM. This was markedly reduced after treatment with UDG. UDG treatment specifically suppressed the formation of artefacts in FFPE DNA and did not affect the detection of true KRAS codon 12 and EGFR exon 19 and 20 mutations. In conclusion, this study reports for the first time that uracil lesions due to cytosine deamination is one of the major causes of artefacts in FFPE DNA and the use of uracil-DNA-glycosylase that removes uracil lesions on DNA can lead to a significant reduction of artefacts. This finding will thus greatly improve biomarker studies using DNA samples derived from FFPE tissues, which has immediate implications especially to diagnostic settings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2098. doi:1538-7445.AM2012-2098
Abstract 2099: RNA from brush cytology to noninvasively screen for oral squamous cell carcinoma
Authors: Kolokythas Antonia , Schwartz Joel L. , Panda Suchismita , Bosman Mitchell J. , Pytynia Kristen , Yeung Alexander , Adami Guy R.
Published: August 2012

Much effort is invested in defining gene expression changes that can be monitored in surgical and biopsy samples of oral lesions to identify oral squamous cell carcinoma (OSCC), a cancer that strikes over 30000 people in the United States each year. There is an extensive list of genes shown to be differentially expressed in surgically obtained oral tumor tissue versus normal, non-malignant, tissue. These have been catalogued over the years and have the potential to inform OSCC characterization. At the same time noninvasive detection and diagnosis of OSCC is a goal that is being explored using a number of methods including brush oral cytology. Using RNA from oral cells obtained with brush cytology we tested levels of mRNAs of 22 genes shown to be differentially expressed in surgically obtained OSCC and normal tissue. Of these 10 showed differential expression in our training set of 12 tumor versus 18 non-malignant lesion samples all from tobacco users. Using Support Vector Machines an algorithm was developed that provided class prediction based on a subset 5 of these mRNAs. With the exception of one case, cross validation during training revealed that this OSCC classifier showed greater than 92% accuracy in differentiating OSCC from non-malignant oral lesions. We will report on its accuracy for correctly identifying OSCC samples in a validation set of RNA from brush cytology samples from an independent set of samples. This test of this methodology using RNA from brush cytology in identifying OSCC highlights the possibility of developing an RNA based classifier from brush cytology to screen for OSCC and perhaps pre-malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2099. doi:1538-7445.AM2012-2099
Abstract 2106: Long non-coding RNA signatures for melanoma detection in humans
Authors: Reilly Christopher , Perera Rochelle A. , Mazar Joseph , Perera Ranjan J.
Published: August 2012

Melanoma, an aggressive carcinoma that originates in the basal layer of human epidermis, has the highest mortality rate of skin cancers in industrialized countries. Unfortunately, current treatments for metastatic melanoma provide mostly palliative benefits to only a small proportion of patients. Despite the obvious need for improvement in assessment and treatment of metastatic melanoma, the intricate mechanisms underlying melanoma transformation remain unresolved and poorly understood. Thus, there is considerable interest in understanding the molecular basis of initiation and progression of human melanomas. Recent evidence indicates that long non-coding RNAs (lncRNAs), a subclass of noncoding RNA (ncRNA), are associated with carcinogenesis in melanoma as well as several other human cancers. The elucidation of the molecular function of these RNA species promises to bridge gaps in our knowledge of melanoma development in humans. The expression of selected lncRNAs (CR618740 and AF091731) in a panel of melanoma cell lines (pigmented and non-pigmented), melanocytes, and keratinocytes were assayed by DNA microarray (Ncode arrays from Lifetechnologies) and further confirmed by real-time polymerase chain reaction (qRT-PCR). Next, cell lines results were tested with patient samples. Our results indicate that the differential expression of lncRNAs is an indicator of the development of melanoma, and dysregulated expression of these markers may be involved in the transition of melanocytes to melanoma. Furthermore, the presence of significant correlation between melanoma cell line characteristics (stage, cell phenotype, types of mutations and others) and lncRNA expression data may imply novel causal connection between lncRNA expression and melanoma development. Importantly, this work will investigate the new possibility that lncRNAs play an essential role in cellular regulation and represent a potential platform for melanoma progression. Thus, unraveling the differential lncRNA expression in melanoma could initiate novel understanding of melanocyte homeostasis, tissue organization, and melanoma genesis in humans.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2106. doi:1538-7445.AM2012-2106
Abstract 2108: Prevalence of mutations in MSH6, PMS1 and PMS2 gene in Brazilian families with Lynch syndrome
Authors: Silva Felipe C. , Torrezan Giovana T. , Lisboa Bianca G. , Santos Erika M. , Gomy Israel , Rossi Benedito M. , Ferreira Fabio O. , Carraro Dirce M.
Published: August 2012

Background: Lynch syndrome (LS) is an autosomal dominant disorder predisposing to colorectal cancer and increased risk for cancers of the stomach, small intestine, hepatobiliary system, kidney, ureter, ovary, and sebaceous tumors. Germline mutations in the MMR genes MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), post- meiotic segregation increased 1 (PMS1) or post- meiotic segregation increased 2 (PMS2), are known to cause LS. While mutations in MLH1 and MSH2 gene, account for 70% - 80% of all Lynch syndrome colorectal cancers, the involvement of the MSH6, PMS2 and PMS1 gene is continually rising. Altogether, MSH6, PMS1 and PMS2 mutations account for 5-20% of kindreds in which MSH2 and MLH1 mutations are excluded. Objective: The aim of the study was evaluate the frequency of mutations in MSH6, PMS1 and PMS2 gene. Methods: Seventy one unrelated patients without MLH1/MSH2 mutations were eligible for this study. Genomic DNA was obtained from peripheral blood and primers were designed within introns of all exons of MSH6, PMS1 gene. In order to avoid the amplification of pseudogenes for the PMS2 gene, a set of four primer pairs were design. Results: Out of 71 patients, 20 fulfilled the Amsterdam criteria and other 51 fulfilled the Bethesda guideline. Four novel pathogenic mutations were detected, three in MSH6 and 1 in PMS2. In addition, 7 variants of uncertain significance were encountered, in which two of them were previously described as pathogenic. No mutations were found in PMS1 gene. Conclusion: Our data shows that 8.5% (6/71) of the individuals have mutations in MSH6 and PMS2 gene and these results strongly suggest that MSH6 and PMS2 should not be excluded from the MMR screening in Brazilian families suspected for Lynch syndrome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2108. doi:1538-7445.AM2012-2108
Abstract 211: Ubiquitin-proteolytic regulation of hematopoietic stem cell expansion
Authors: Lee Jennifer , Shieh Jae-Hung , Zhang Jianxuan , Liu Liren , Morrone Giovanni , Moore Malcolm A.S. , Zhou Pengbo
Published: August 2012

Direct transduction of the homeobox protein HOXB4 promotes the proliferation of hematopoietic stem cells (HSCs) without induction of leukemogenesis, but requires frequent administration to overcome its short protein half-life. We demonstrate here that the CUL4A ubiquitin ligase regulates the post-translational stability of multiple HOX proteins, including HOXB4, and we defined the degron of HOXB4 required for CUL4A-mediated degradation. Ectopic expression of degradation-resistant HOXB4 in myeloid progenitor cells confers a growth advantage over wild-type HOXB4-expressing cells. Finally, direct transduction of recombinant degradation-resistant HOXB4 protein to human adult HSCs significantly enhanced their maintenance in a more primitive state both in vitro and in vivo compared to transduction with wild-type HOXB4 protein. Our studies demonstrate the feasibility of engineering a stable HOXB4 variant to overcome a major technical hurdle in the ex vivo expansion of adult HSCs and early progenitors for human therapeutic use.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 211. doi:1538-7445.AM2012-211
Abstract 2111: Characterization of current and novel biomarkers of bladder cancer
Authors: Fowler Elizabeth , Woolworth Julie , Shao Yuan , Sobolesky Philip , Keane Thomas , Wolff Daynna , Watson Dennis , Moussa Omar
Published: August 2012

Bladder cancer is one of the most common cancers in industrialized countries. It is reported that 3.51 billion dollars are spent each year on bladder cancer treatment making it the most expensive cancer to treat on a per patient basis. Approximately 80% of bladder cancer patients have at least one reported recurrence thereby prompting meticulous lifetime screening and surveillance by health care professionals. Therefore, careful evaluation of our institution's current fluorescent in situ hybridization surveillance tools in addition to investigation of novel biomarkers is imperative. Our investigation of novel biomarkers focused on apoptosis regulating genes which are often unbalanced in cancerous tissues. Our institution recently adopted BioView, an automated FISH capture/analysis system, to replace the manual protocol. Validation and assessment of the automated system for fluorescent in situ hybridization (FISH) interpretation to detect urothelial cancer was determined using patient cases to evaluate concordance, overall positivity, interpretative time spent, sensitivity, and specificity. To evaluate novel biomarkers, mRNA from patient samples was reversed transcribed to cDNA then utilized for biomarker quantification by RT-PCR. In addition, immunohistochemistry was performed on the most promising gene to further confirm its clinical usefulness. The automated BioView capture/ detection system does prove to be more time efficient and accurate when addressing all parameters described. In evaluation of novel biomarkers, API5, BIRC3, BIRC4, SURVIVIN, and BFAR provided the most promising data with sensitivity and specificity percentages all ranging from 83% to 97%. From this study of bladder cancer markers, we focused on API5 and its role in bladder cancer initiation and progression. API5 is strongly expressed early in tumorigenesis. Our data confirm that the automated FISH capture/detection system improves laboratory efficiency and accuracy. Furthermore, our data indicate that apoptotic regulators are promising biomarkers for clinical use. In addition, API5 is expressed early in tumorigenesis, which makes it an ideal early diagnostic marker. We have noticed increase in the level of API5 protein associated with tumor grade and stage which also suggest that API5 might be a potential prognostic marker for bladder cancer. In conclusion, detection of apoptosis regulating genes is a promising diagnostic and prognostic approach to aid our screening and surveillance tools.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2111. doi:1538-7445.AM2012-2111
Abstract 2111A: Molecular genetics of non-alcoholic fatty liver disease
Authors: Shi Ivy , Sakaguchi Takuya
Published: August 2012

Non-alcoholic fatty liver disease (NAFLD) is a widespread condition of growing concern. It affects up to 25% of people in the United States, and is caused by the buildup of lipids in the liver. NAFLD may lead to hepatocellular carcinoma, the 5th most common cancer in the world, and the 3rd most common cause of death from cancer. Steatosis is the earliest stage in the progression of NAFLD. It is characterized by the deposition of triglycerides as lipid droplets in the cytoplasm of hepatocytes. Although not usually lethal alone, it may develop into nonalcoholic steatohepatitis, and develop further into cirrhosis and ultimately hepatocellular carcinoma. A mutation in zebrafish is created that completely obstructs the guanine monophosphate synthase. The mutants show a phenotype of steatosis. We hypothesize that genes involved in de novo lipogenesis, lipid oxidation, or lipid transportation are altered in the mutants. To test our hypothesis, we conducted gene expression analysis using qPCR. The data indicates that the genes involved in de novo lipogenesis had no significant change. However, the genes involved in lipid oxidation were all significantly downregulated. This downregulation indicates that inadequate lipid metabolism is responsible for the buildup of lipids and steatosis. The genes involved in lipid transportation showed a downregulation trend. It might be a result of steatosis as the liver takes in less fat. Overall, the data suggests that the mutant cell is unable to metabolize enough lipids through oxidation, causing lipid buildup in the liver.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2111A. doi:1538-7445.AM2012-2111A