bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 179: Impact of diet-induced obesity on inflamatory pathways during prostate cancer progression in Hi-Myc mice.
Authors: Blando Jorge M. , Kiguchi Kaoru , Fernandez Irina , DiGiovanni John
Published: August 2014

We have previously reported that diet induced obesity (DIO) enhanced prostate cancer progression and associated inflammation in the ventral prostate of the Hi-Myc mice (Blando et al, CAPR, 2011). Here, we further investigated changes in inflammatory genes as well as cellular changes associated with DIO effects on prostate cancer progression in the Hi-myc mouse model. Initial studies were conducted to look at specific cell populations that are present in the ventral prostate (VP) of Hi-Myc mice on the 30% CR, overweight control (AIN76A) and DIO diets (60 Kcal% fat). Staining for perilipin revealed that adipocytes were restricted to the periphery of the VP of Hi-Myc mice on the 30% CR diet. In contrast, adipocytes could be seen infiltrating the VP of Hi-Myc mice on the overweight control diet and especially in mice on the DIO diet. In this regard, many of the glands in the VP were completely surrounded by adipocytes. Double IF staining was performed for both perilipin and CD3 (T-lymphocytes) or perilipin and RM0029-11H3 (macrophages). Few if any inflammatory cells were seen in VP of Hi-Myc mice on the 30% CR diet. In contrast, adipocytes and inflammatory cells (both T-cells and macrophages) were seen in close proximity to adipocytes that had infiltrated into the VP in the overweight control group and this was particularly prominent in the DIO diet group. Aditionally, staining for B-cells also showed an increased number in the DIO groups as compared to the control and CR diet groups. Sections of VP from Hi-Myc mice on the different diets were also stained for both the IL-23 receptor (IL-23R) and Cox-2. IL-23R expression was dramatically upregulated in the epithelial cells of the VP from Hi-Myc mice on the DIO compared to the other diet groups. Cox-2 expression was also dramatically upregulated in VP of Hi-Myc on the DIO diet. This finding is consistent with the high level of NFκB activation seen in prostate tissues for the same mice and diet group. The VP from mice on DIO diets also showed an increase in the staining of tissue specific cancer stem cell (CSC) markers (SCA-1 and CD49f) when compared to VP from mice on the overweight control and 30% CR diets. In summary, changes in the levels of IL-23R and COX-2 and the increased presence of inflammatory cells together with changes in the adipose tissue, suggest that the tumor microenvironment may play an important role in DIO effects on prostate cancer progression in Hi-Myc mice.

Citation Format: Jorge M. Blando, Kaoru Kiguchi, Irina Fernandez, John DiGiovanni. Impact of diet-induced obesity on inflamatory pathways during prostate cancer progression in Hi-Myc mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 179. doi:10.1158/1538-7445.AM2013-179
Abstract 1791: Tristetraprolin downregulates AHRR expression through mRNA destabilization.
Authors: Lee Hyun Hee , Kim Won Tae , Kim Dong Hee , Leem Sun-Hee
Published: August 2014

Background. The aryl hydrocarbon receptor repressor (AHRR) inhibits the transcription activity of the aryl hydrocarbon receptor (AHR) by competing for dimerization with the AHR nuclear translocator (ARNT) and subsequently binding to XRE. Interestingly, the AHRR has two AU-rich elements (AREs) in its 3’UTR which relates to the post-transcriptional regulation by ARE-binding proteins including Tristetraprolin (TTP), BRF1, HuR and so on. In previous study, we determined that TTP plays a critical role in the decaying of VEGF, LATS2, cIAP2, COX2 and Pim-1 through binding these AREs in 3’ UTR.

Methodology/principal Findings. We investigated the post-transcriptional regulation of the AHRR expression by TTP. Our results show that the expression level of AHRR is inversely correlated with TTP expression in human breast cancer cell lines using the real-time PCR and western blot. The overexpression of TTP decreased the expression level of AHRR and inhibited the proliferation of MDA-MB435 human breast cancer cells. On the contrary, the treatment with a small interfering RNA against TTP increased the expression of AHRR mRNA and protein. AHRR mRNA contains two AREs within the 3’UTR and TTP destabilized a luciferase mRNA containing AHRR ARE. Moreover, RNA electrophoretic mobility shift assay revealed that TTP binds directly to the ARE of AHRR mRNA.

Conclusions/Significance. These studies demonstrated that the TTP expression is inversely correlated with AHRR expression in several human breast cancer cell lines using the real-time PCR and western blot. We suggest that TTP acts as a negative regulator of the AHRR gene expression and it may have an effect on tumor development through inducing AHR activation and the expression of tumor suppressors containing XRE in human breast cancer cells.

Citation Format: Hyun Hee Lee, Won Tae Kim, Dong Hee Kim, Sun-Hee Leem. Tristetraprolin downregulates AHRR expression through mRNA destabilization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2013-1791
Abstract 1798: The human mammary circadian transcriptome.
Authors: Combs Joseph , Mandichak Tracy , Ferree Elizabeth , Hoffman Aaron E.
Published: August 2014

The circadian rhythm, a phenomenon present in all of Eukaryota and in some members of Prokaryota, describes the processes within an organism that fluctuate throughout a daily cycle. Within peripheral tissues, clock gene expression is organized into a roughly 24-hour period by an elaborate feedback loop mechanism developed around a core set of circadian genes. In addition to maintaining rhythmicity within the molecular clock, key circadian genes such as CLOCK and ARNTL1 are also transcription factors that regulate the expression of a number of additional transcripts; the so-called clock-controlled genes (CCGs). This transcriptional control forms the backbone of circadian rhythmicity and is critical for the maintenance of proper cellular function in a variety of physiological processes, ranging from cell cycle regulation to cellular metabolism. Despite the importance of this regulatory clockwork system, little is known about the extent of molecular circadian control in human peripheral tissues. In fact, the majority of our knowledge of molecular oscillatory systems is derived from studies of model organisms which consistently demonstrate that outside of the core circadian genes; there is virtually no overlap in the compendium of CCGs across tissue types. Based on this critical observation, it is reasonable to expect that if CCGs vary considerably across tissues within the same organism, there may also be considerable heterogeneity across organisms, even within the same tissue type. Here we use next-generation sequencing analyses to provide the first circadian transcriptome in pre-stasis non-immortalized primary human mammary epithelial tissue. Using the non-parametric JTK_CYCLE algorithm, we found that approximately 6% of all expressed genes within this tissue are CCGs. We also find considerable evidence for circadian control in RNA processing events, including splice isoform usage and alternative polyadenylation, and a pathway analysis of all CCGs indicates circadian involvement in multiple signaling and growth pathways. These results demonstrate, for the first time, that a significant and important proportion of the human mammary transcriptome is regulated by the circadian system. Given the importance of circadian disruption in the pathophysiology of a range of human disorders, from metabolic disease to breast cancer, we believe that categorization of the circadian rhythm in human peripheral tissue will lay an important foundation for future breakthroughs in disease prevention, as well as in the development of novel therapeutic strategies.

Citation Format: Joseph Combs, Tracy Mandichak, Elizabeth Ferree, Aaron E. Hoffman. The human mammary circadian transcriptome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1798. doi:10.1158/1538-7445.AM2013-1798
Abstract 1800: Expression analysis of PI3K pathway genes in head and neck cancer.
Authors: Buyru Nur , Ekizoglu Seda , Karaman Emin , Enver Ozgun
Published: August 2014

Head and neck cancer (HNC) is a heterogenous group of malignant tumors which includes neoplasias of the oral cavity, the pharynx, the larynx and paranasal sinuses. Squamous cell carcinomas comprise more than 95% of all head and neck cancers. The vast majority of HNC is related to exposure to carcinogens, including tobacco, alcohol, betel nut, and sexually transmitted viral pathogens such as human papilloma viruses (HPV). However, only a small fraction of tobacco and alcohol users develop the disease suggesting that genetic factors may play a key role in HNC.

Increasing evidence indicate that mTOR is a central regulator of cell growth, proliferation, survival and dysregulation of the PI3K/Akt/mTOR signaling pathway that occurs in many tumors including head and neck cancer. The TSC complex (TSC1/TSC2) negatively regulates mTOR by inhibiting a small GTP binding protein, Rheb, considered to be the direct activator of mTOR. On the other hand, the Ras protein upregulates PI3K signaling which is an upstream effector of mTOR. When PI3K activates Akt, the dual function phosphatase PTEN negatively regulates it. Human cancers frequently harbor activating mutations in the p110 catalytic subunit of PI3K or genomic amplification of Akt. Activated Akt also promotes survival under conditions of cellular stress.

In this study we investigated the expression of the critical genes (mTOR, K-ras, PI3KCA, TSC1, RHEB, PTEN and AKT1) acting in this pathway. For this purpose, we analyzed expression levels of these genes in 49 matched HNC tumor and non-cancerous tissue samples by quantitative RT-PCR. We observed a significant downregulation of the mTOR (p=0.005), TSC1 (p=0.001), and PTEN (p=0.000) mRNA expressions. On the other hand, a slight decrease was also observed in K-ras, Akt1 and PI3KCA mRNA levels in tumor tissues compared to the non-cancerous ones. However, the difference in the expression levels of these genes between the two groups were not statistically significant. Whereas we didn't observe any difference in Rheb mRNA expression level in tumor tissue when compared to the non-cancerous tissue samples. mTOR, TSC1, and PTEN mRNA expression levels were decreased 0.48, 0.44 and 0.32 % in HNC tumor tissue samples compared to non-cancerous tissue samples, respectively .

Our result indicate that, the PI3K pathway has an important function in HNC progression with the contribution of more than one gene of this pathway.

Citation Format: Nur Buyru, Seda Ekizoglu, Emin Karaman, Ozgun Enver. Expression analysis of PI3K pathway genes in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1800. doi:10.1158/1538-7445.AM2013-1800

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1811: Cancer/testis antigens: Comprehensive expression analysis and correlation with survival in glioblastoma.
Authors: Freitas Marcelo , Biassi Thaís P. , Malheiros Suzana M.F , Stávale João N. , Zamunér Fernando T. , Begnami Maria D.F.S , Soares Fernando A. , Vettore André L.
Published: August 2014

Glioblastoma (GBM) is a highly invasive and aggressive tumor which confers a dismal prognosis despite advances of current therapy. Cancer vaccines have been utilized as treatment of other tumors and might be an option for brain tumors. Cancer/testis (CTA) genes represent promising targets for immunotherapeutic approaches. They are expressed in tumors and expression in normal tissues is exclusively or preferentially restricted to testis. The expression of 31 CTA genes was investigated in 48 glioblastomas and 5 normal brain samples using RT-PCR. A tissue microarray was made and expression of a CTA was detected by immunohistochemical staining. Among the genes with no expression in normal brain CTA1 (57%), CTA2 (54%), CTA3 (44%) and CTA4 (15%) were the most expressed in glioblastomas. At least one of these four selected CTA genes was found expressed in 86% of the GBM cases. Coexpression of two, three and four CTA genes occurred in 25%, 17% and 5% of the examined GBM cases, respectively. CTA4 protein expression was identified in 13% (12/92) of GBM and was negative in all anaplastic astrocytomas (0/15), diffuse astrocytomas (0/48), pilocytic astrocytomas (0/38) and normal brain samples (0/40) evaluated. We found that CTA2-positive GBMs and tumors expressing 3-4 CTA genes presented a significantly better outcome (P = 0.032; hazards ratio = 0.53; 95% CI; 0.28 to 1.0 and P = 0.017, hazards ratio = 0.36; 95% CI, 0.17 to 0.74; respectively). Furthermore, multivariate survival analysis revealed that coexpression of 3-4 CTA genes (P = 0.044, hazards ratio = 0.3, 95%CI = 0.093-0.963), radiotherapy (P = 0.010, hazards ratio = 0.16, 95%CI = 0.04-0.65) and chemotherapy (P = 0.001, hazards ratio = 0.10, 95% CI = 0.03-0.41) remained as independent predictors of overall survival. In conclusion, CTAs are potential targets for immunotherapy in patients with GBM, 86% of the cases examined expressed at least one of four selected CTA genes. The presence of 3-4 CTA genes was associated with better survival, suggesting that these genes might elicit an immune response against GBM. Supported by FAPESP(Grant 2010/20218-2 and 2011/15118-1).

Citation Format: Marcelo Freitas, Thaís P. Biassi, Suzana M.F Malheiros, João N. Stávale, Fernando T. Zamunér, Maria D.F.S Begnami, Fernando A. Soares, André L. Vettore. Cancer/testis antigens: Comprehensive expression analysis and correlation with survival in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1811. doi:10.1158/1538-7445.AM2013-1811
Abstract 183: Beta-endorphin neuron transplants suppress mammary tumor growth and progression: role of immune cells.
Authors: Sarkar Dipak K. , Zhang Changqing , Murugan Sengottuvelan , Boyadjieva Nadka , Ortigüela María
Published: August 2014

We have recently identified that a set of hormone secreting nerve cells in the hypothalamus, called beta-endorphin (BEP) neurons, have the ability to prevent the growth, progression and metastasis of mammary and prostate tumors in laboratory animals. How these neuronal cells prevent tumor cell growth and progression is not apparent. In order to better understand the mechanism by which endorphin neurons control tumor growth, we determined the growth and clearance of breast cancer derived MADB106 cells in Fisher rats, which are immune intact, and in athymic Nude rats, which are T cells deficient. Adult female Fischer rats or Nude rats were transplanted with either BEP neurons or cortical neurons (control) in the hypothalamus for 4 weeks and then inoculated with MADB106 cells in the jugular vein or under the skin in the back. These animals were sacrificed after 6 weeks to determine the volume of the tumor at the site of inoculation for determining the growth of tumors and the number of other tumors at the distant sites for determining tumor metastasis. Tumors were also collected for histopathology. Blood samples were collected and used for measurements of cytokines and immune cells. In Fischer rats, after tumor inoculation, animals with control cell transplants had tumor colonization in the lung and some on the skin at the site of cell injection. However, BEP neurons treated Fischer rats had no tumor colonization in the body. In the Nude rats, tumor colonization occurred both at the site of injection and at the distant site under the skin as well as in the lung of control transplanted rats. In BEP neurons transplanted Nude rats, tumor colonization only occurred at the site of inoculation. The mean volume of tumor at the inoculation site was smaller in BEP neurons transplanted rats than in control cells transplanted rats. In BEP neurons transplanted rats, corticosterone and inflammatory cytokines levels were lower, whereas anti-inflammatory cytokines and chemokines levels were higher. In addition, NK cell and macrophage cell numbers and their cytolytic functions in peripheral blood of BEP neurons transplanted rats were elevated. These data suggest that the antitumor action of BEP neuron transplants is due to the activation of primarily NK cells and macrophages and also in lesser extent T cells. The data also identify the potential use of BEP cell therapy for cancer prevention. (Supported by R37 AA08757)

Citation Format: Dipak K. Sarkar, Changqing Zhang, Sengottuvelan Murugan, Nadka Boyadjieva, María Ortigüela. Beta-endorphin neuron transplants suppress mammary tumor growth and progression: role of immune cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 183. doi:10.1158/1538-7445.AM2013-183
Abstract 1838: nc886, a putative tumor suppressive non-coding RNA in gastric cancer.
Authors: Kim Seon-Young , Park Jong Lyul , Kim Yong Sung , Lee Kwang Soo , Jeon Sung Ho , Lee Yong Sun
Published: August 2014

nc886, a novel type of non-coding RNA, was originally recognized as a microRNA precursor (pre-miRNA) or a vault RNA (vtRNA), but it has been shown to be barely processed into mature microRNAs and be mostly dissociated from the vault complex. So far, nc886’s best studied role is as a cellular RNA ligand and regulator of PKR (Protein Kinase RNA-activated). When nc886 is suppressed, PKR is activated and this activation leads to apoptosis through the canonical PKR-eIF2α cell death pathway. Given that the expression of nc886 was found to be suppressed in a variety of cancer cells, nc886/PKR's such role is thought to be critical in eliminating pre-cancerous cells during certain stages of tumorigenesis. Thus, nc886 plays a sentinel role against tumorigenesis by controlling PKR activity in some cells.

We have found that nc886 is decreased also in gastric cancer. Our measurement of nc886 in 88 specimens from gastric cancer patients indicates that nc886 tends to be decreased in tumors relative to the adjacent normal tissues. Also, DNA methylation of the nc886 locus exhibits an anti-correlation to nc886 level, and therefore epigenetic silencing is likely to be the mechanism for the decrease of nc886 in gastric cancer. Moreover, hypermethylation of the nc886 promoter region is associated with poor survival of gastric cancer patients. All our data indicates a putative tumor suppressive role for nc886 and the possibility of a clinical application for nc886 as a prognostic marker for gastric cancer.

Citation Format: Seon-Young Kim, Jong Lyul Park, Yong Sung Kim, Kwang Soo Lee, Sung Ho Jeon, Yong Sun Lee. nc886, a putative tumor suppressive non-coding RNA in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1838. doi:10.1158/1538-7445.AM2013-1838
Abstract 1841: Characterizing long non-coding RNAs located within a replication origin at the human DBF4 locus.
Authors: Hughes Shawn , Kylie Kathleen , Romero Julia , Lee Hoyun
Published: August 2014

Faithful replication of the entire genome only once per cell cycle is crucial to cell homeostasis. The importance of proper replication is manifested by the presence of a large library of strict regulatory mechanisms that a cell employs to govern entry into the cell cycle. Failure to regulate this “licensing” process may result in a state of aneuploidy which is a driving force in cancer development. Besides the licensing proteins, the precise mechanism of how a cell chooses or activates replication initiation points is still poorly understood in mammalian cells. We have been studying the replication origin located at the promoter region of the human DBF4 gene. This origin contains two replication initiation zones that fire in opposite directions sequentially in a time-dependant manner, which we've termed it asymmetrical bidirectional replication (ABR). We have recently discovered that the DBF4 origin region contains a unique distribution and modifications of histones, which appears to be relevant to the regulation of replication initiation. In addition, we also found high activities of long non-coding RNA (lncRNA) transcription up- and down-stream of the DBF4 origin. Our preliminary data suggest that the active transcription of the lncRNAs at this locus may have a regulatory function on the chromatin conformation, transcription and DNA replication. We are currently carrying out experiments to gain a better understanding of the mechanism how lncRNA transcription is regulated in the context of replication initiation and DBF4 transcription.

Citation Format: Shawn Hughes, Kathleen Kylie, Julia Romero, Hoyun Lee. Characterizing long non-coding RNAs located within a replication origin at the human DBF4 locus. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1841. doi:10.1158/1538-7445.AM2013-1841
Abstract 1849: A novel gene-pathway regulating of microRNA mediates resistance to trastuzumab forgastric cancer.
Authors: Eto Kojiro , Watanabe Masayuki , Iwatsuki Masaaki , Baba Hideo
Published: August 2014

Background: Recently, the efficacy of trastuzumab is indicated in patients with unresectable or recurrent HER2 positive gastric cancer from the results of ToGA trial, and trastuzumab administration was to be done in patients with gastric cancer. However, resistance to trastuzumab has become a problem in breast cancer. MicroRNAs (miRs) are involved in regulating multiple cellular pathways, including cell proliferation, differentiation, and chemotherapy resistance, and thus may function as oncogenes or tumor suppressing genes. The aim of current study is to clarify the miR-gene pathway regulating resistance to trastuzumab. Method: Using 6 gastric cancer cell lines, we examined the following items in vitro. 1. We examined the status of HER2 protein expression by western blotting. 2. We focused on miR-21 and its target gene PTEN and evaluated the expression of miR-21 and PTEN in HER2 positive gastric cancer cell lines by real time PCR and western blotting. 3. We examined the expression of PTEN and downstream gene to clary the target gene of miR-21 by over or down regulation of miR-21. 4. We investigated whether miR-21-PTEN pathway can affect the resistance of trastuzumab. Result: 1.We confirmed the status of HER2 in gastric cell lines. 2. The endogenous expression of PTEN and miR-21 showed inverse correlation in each cell lines. 3. The suppression of miR-21 increased PTEN expression with downregulation of P-Akt, persisting HER2 expression. 4. Overexpression of miR-21 can induce the resistance of trastuzumab, and suppression of miR-21 can restore the sensitivity of trastuzumab. Conclusion: Our current study revealed that miR-21-PTEN pathway can regulate the sensitivity of trastuzumab in HER2-positive gastric cancer cell lines. These findings suggest that this pathway can be crucial clue to the mechanism of resistance to trastuzumab in gastric cancer. To elucidate the mechanism of resistance to trastuzumab can lead to the development of individualized treatment.

Citation Format: Kojiro Eto, Masayuki Watanabe, Masaaki Iwatsuki, Hideo Baba. A novel gene-pathway regulating of microRNA mediates resistance to trastuzumab forgastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1849. doi:10.1158/1538-7445.AM2013-1849
Abstract 1850: BRCA1-regulating microRNAs, miR-146a and miR-638, are overexpressed in triple negative breast cancer tumors.
Authors: Zavala Valentina , Fernandez Wanda , Cruz Adolfo , Segovia Laura , Camus Mauricio , Carvallo Pilar
Published: August 2014

microRNAs are non-coding RNA sequences of about 22 nucleotides long that participate in post transcriptional regulation of mRNAs. Its deregulation has been associated to breast cancer development and treatment resistance. Triple negative breast tumors are characterized by its difficult treatment due to lack of estrogen and progesterone receptors and ERBB2. In addition, a high proportion do not express BRCA1; a breast cancer high susceptibility gene. BRCA1 expression can be regulated by different mechanisms as we have confirmed in our laboratory, such as methylation of its gene promoter, mutations and deletions due genomic rearrangements. Although there are tumors that do not present any of these abnormalities, they express low nuclear BRCA1 levels or they do not express the protein at all. This is why analyzing levels of microRNAs that are shown to regulate BRCA1 mRNA expression is an interesting additional mechanism to be evaluated. There is experimental evidence that miR-146a and mir-638 regulate BRCA1 expression. We therefore analyzed miR-146a and miR-638 in triple negative breast cancer tumors (n=41). Briefly, total RNA was extracted from formalin-fixed tumors. Then, both microRNAs were evaluated using qPCR TaqMan assay and compared with BRCA1 expression previously determined by immunohistochemistry. Our results showed overexpression of miR-146a in 68% of the analyzed tumors and miR-638 in 82%. Among those tumors presenting abnormal BRCA1 expression (n=24), 50% showed an increase of miR-146a and 75% presented miR-638 overexpression. Both microRNAs showed a discrete overexpression in tumors with normal levels of BRCA1 (n=16). Finally, this data suggest that microRNAs might regulate BRCA1 expression in triple negative tumors.

Citation Format: Valentina Zavala, Wanda Fernandez, Adolfo Cruz, Laura Segovia, Mauricio Camus, Pilar Carvallo. BRCA1-regulating microRNAs, miR-146a and miR-638, are overexpressed in triple negative breast cancer tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2013-1850
Abstract 1851: Resveratrol impairs GLUT-1 mediated glucose uptake in ovarian cancer cells .
Authors: Gwak HyeRan , No Jae Hong , Song Yong Sang
Published: August 2014

Resveratrol(RSV), a phytoalexin found in red wine, has drawn interest due to its anticancer effect through the modulation of cancer cell metabolism in in vitro and in vivo studies. Increased uptake of [18F] fluorodeoxyglucose (FDG) using positron emission tomography (PET) has been found to be effective diagnostic tool for ovarian cancer. Thus, the manipulation of the metabolic derangement could be an effective therapeutic target for ovarian cancer. In this study, we found that RSV inhibited glucose uptake via inhibition of Glut1 translocation to plasma membrane and induced apoptosis irrespective of p53-status, using four ovarian cancer cell lines, PA-1 (p53-wild), OVCAR3, MDAH 2774 (p53-mutant) and SKOV3 (p53-null). RSV (50 μM) treatment in ovarian cancer cells decreased glucose uptake in parallel with cell viability. The facilitative glucose uptake by glucose transporter1 (GLUT1) seems to be a primary step in ovarian cancer. Also in previous data, mRNA of GLUT1 was more highly expressed in tumor tissue than in normal tissue in patients with ovarian cancer. However, RSV did not affect the protein levels of GLUT1. To identify translocation of Glut1, we performed immunofluorescence and immunoblotting of total and membrane-bound proteins. GLUT1 translocation to plasma-membrane was found to be decreased. Furthermore, localization of GLUT1 in ovarian cancer was found to be associated with the inhibition of Akt activity by RSV. In conclusion, RSV induced apoptosis by impairing glucose uptake through modulating Akt-GLUT1 signaling axis in ovarian cancer cells.

Citation Format: HyeRan Gwak, Jae Hong No, Yong Sang Song. Resveratrol impairs GLUT-1 mediated glucose uptake in ovarian cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1851. doi:10.1158/1538-7445.AM2013-1851
Abstract 1852: Pyruvate dehydrogenase kinase expression and metabolic changes following dichloroacetate exposure in anoxia in human colorectal cancer cells.
Authors: Ho Nelson , Coomber Brenda L.
Published: August 2014

Introduction: While glycolysis is a suboptimal metabolic strategy used by cells under hypoxic conditions, many cancer cells preferentially utilize glycolysis for energy production in the presence of abundant oxygen, a phenomenon known as the “Warburg Effect”. Dichloroacetate (DCA) is a small molecule metabolic modulator that shifts cellular metabolism to glucose oxidation by inhibition of pyruvate dehydrogenase kinase (PDK), an enzymatic inhibitor of pyruvate dehydrogenase (PDH). Furthermore, DCA has been shown to induce apoptosis in cancer but not normal cells. Our previous work showed that some human colorectal cancer (CRC) cells are protected from anoxia-induced apoptosis upon exposure to DCA, through as yet unclear mechanisms.

Hypothesis: It is hypothesized that differential expression of PDK isoforms will correlate with the ability of different cancer cells to withstand the apoptotic effects of DCA in anoxia.

Methods: SW480, LS174T (CRC) and IEC-18 (normal intestinal epithelial) cell lines were exposed to 10 mM DCA in either normoxic (20% O2) or anoxic (< 0.1% O2) conditions for 24 hours. Following treatment, expression of different PDK isoforms was determined through qRT-PCR and western blotting. Phosphorylation of PDH at Ser293 and Ser300 was assessed through western blotting. Glucose consumption and lactate production was determined using a colorimetric-based assay. Changes in mitochondrial function were quantified through Mitotracker staining and subsequent flow cytometry. Cell viability was assessed using the neutral red assay.

Results: In all three cell lines: A) PDK isoforms 1 and 3 are highly expressed relative to isoforms 2 and 4, B) DCA and/or anoxic treatment induced a greater effect on PDH phosphorylation at Ser300 than Ser293, and C) DCA had minimal effect on glucose consumption in either normoxia or anoxia.

Conclusions: Altered PDK expression and PDH activation may not fully explain the differences in resistance to DCA-induced apoptosis observed in human CRC cells. Investigating the mechanisms by which certain cancer cells are protected from DCA-induced apoptosis will lead to a better understanding of cancer metabolism and thus provide better therapeutic opportunities.

Citation Format: Nelson Ho, Brenda L. Coomber. Pyruvate dehydrogenase kinase expression and metabolic changes following dichloroacetate exposure in anoxia in human colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1852. doi:10.1158/1538-7445.AM2013-1852
Abstract 1856: Preclinical study for statins as anticancer drug.
Authors: Jiang Pengfei , Mukthavavam Rajesh , Chao Ying , Nomura Natsuko , Fogal Valentina , Pastorino Sandra , Summit Ila , Kesari Santosh
Published: August 2014

Statins are a class of drug that inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase that is the rate-limiting enzyme of cholesterols synthesis. Now, statins are among the most prescribed drug used for treatment of hypercholesterolemia by lower down the serum cholesterol concentration and it is also a major means to preventing and reducing the risk for cardiovascular diseases. In addition to prevent and treat cardiovascular diseases, the emerging evidences indicated the statins usage has a number of beneficial effect such as anti-inflammation and lowering down the risk of cancer. To investigate the anti-cancer effect statins drugs that have been approved to clinical usage we evaluated the inhibition of breast cancer and GBM cell line growth. Statins are also potent inhibitors to stem cell-like primary GBM cells freshly isolated from patients. Unexpected, statins treatment induces strong cell autophagy death signals as weak if not at all cellular apoptosis. Tumor cells can be rescued after statin treatment by adding intermediated product of cholesterol synthesis (mevalonate and GGPP), it indicates that the target of the statins is specifically related the cholesterols synthesis pathway. Knock-down the expression of geranylgeranyl pyrophosphate synthetase-1 (GGPS-1), another key enzyme in cholesterol synthesis pathway, also stimulated strong cell autophagy and cell death in vitro, also dramatically reduced U87 tumor growth in vivo. In vivo data also showed that directly injection of statin is better than oral administrate to delay GBM tumor growth. This study showed statins are potent anti-cancer drug in vitro and in animal model. These safe and well-tolerate drugs are good candidates for clinical test as cancer chemotherapy reagents.

Citation Format: Pengfei Jiang, Rajesh Mukthavavam, Ying Chao, Natsuko Nomura, Valentina Fogal, Sandra Pastorino, Ila Summit, Santosh Kesari. Preclinical study for statins as anticancer drug. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1856. doi:10.1158/1538-7445.AM2013-1856

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1874: Phosphatidylcholine synthesis as a potential therapeutic target in multiple myeloma.
Authors: Bardeleben Carolyne , Lichtenstein Alan
Published: August 2014

A metabolics screen with 8226 cells treated with AICAr showed that de novo pyrimidine synthesis was inhibited. AICAr -induced apoptosis in 5 different myeloma cell lines. This screen showed a significant decrease in CDP-choline, an intermediate of the Kennedy pathway involved in de novo synthesis of phosphatidylcholine (PC). Phosphatidylcholine is the most predominant phospholipid in mammalian ER membranes. The rate limiting step in this pathway is the conversion of phosphocholine and CTP to CDP-choline via the enzyme CTP:phosphocholine cytidylyltransferase (CCT). Although it is likely that the decrease in CDP-choline is a consequence of pyrimidine starvation, the fact that this pathway showed a significant decrease within 8 hours of AICAr treatment suggested to us that this pathway may have a high flux in 8226 cells and that phosphatidylcholine synthesis may be a relevant target in myeloma cells.

Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. Knocking down of CCT with shRNA in myeloma cell lines also resulted in apoptosis. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy.

Citation Format: Carolyne Bardeleben, Alan Lichtenstein. Phosphatidylcholine synthesis as a potential therapeutic target in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1874. doi:10.1158/1538-7445.AM2013-1874
Abstract 1882: Role of cystathionine beta-synthase in ovarian cancer growth, metastasis and drug sensitivity.
Authors: Saha Sounik , Bhattacharyya Sanjib , Giri Karuna , Lanza Ian R. , Nair K. Sreekumar , Jennings Nicholas B. , Kumar Rakesh , Sood Anil K. , Bhattacharya Resham , Mukherjee Priyabrata
Published: August 2014

Hydrogen sulfide (H2S), the third gasotransmitter, has recently attracted a lot of attention for its cytoprotective action and role in angiogenesis. Dysregulation of the major enzymes which produces H2S namely, cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) have also been implicated in a large number of pathological conditions that range from neurological disorders to cardiovascular diseases. Surprisingly, studies on the role of these enzymes in cancer is scarce, especially, that of CBS. Relapse with a drug resistant and aggressive phenotype is a common occurrence in ovarian cancer with an overall 5-year survival of approximately 30%. Here we report that, CBS, a key enzyme required for sulphur amino acid metabolism, plays a critical role in promoting ovarian cancer growth, metastasis and drug resistance. Overexpression of CBS was observed in a majority of ovarian cancer cell lines utilized in our study compared to normal ovarian surface epithelial cells. Silencing CBS in A2780 cells using siRNA or a CBS-specific inhibitor, aminooxyacetic acid reduced proliferation of ovarian cancer cells by dramatically reducing total glutathione levels, H2S levels and thereby enhancing oxidative stress. Downregulation of CBS reduced activation of NF-kB and upregulated p53 protein levels thereby inactivating the antioxidant network of the cells. A deeper insight into the intracellular localization of CBS by confocal fluorescence microscopy demonstrated a mitochondria specific distribution and silencing CBS caused mitochondrial superoxide accumulation that resulted in decreased mitochondrial respiration, reduced ATP production and increased ADP-to-ATP ratio. Furthermore, silencing CBS, augmented the activity of cisplatin in vitro in A2780 cells and in vivo in an orthotopic model of advanced ovarian cancer. Nanoliposomal delivery of CBS siRNA significantly reduced the number of tumor nodules in a chemoresistant orthotopic mouse model of ovarian cancer. To our knowledge, this is the first study demonstrating the importance of CBS in ovarian cancer progression and drug resistance and therefore is a viable target for future anti-cancer therapies.

Citation Format: Sounik Saha, Sanjib Bhattacharyya, Karuna Giri, Ian R. Lanza, K. Sreekumar Nair, Nicholas B. Jennings, Rakesh Kumar, Anil K. Sood, Resham Bhattacharya, Priyabrata Mukherjee. Role of cystathionine beta-synthase in ovarian cancer growth, metastasis and drug sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1882. doi:10.1158/1538-7445.AM2013-1882
Abstract 1890: Superoxide modulates tumor cell survival in response to hypoxia and metabolic stress: role of p53 status.
Authors: Chavez-Perez Valery , Strasberg-Rieber Mary , Rieber Manuel
Published: August 2014

Background: Cancer cells from solid tumors become metabolically stressed, when nutrients are insufficient within poorly vascularized regions. Metabolic stress resulting from severe deprivation of glucose and serum-derived growth factors can promote cell death, partly through excessive reactive oxygen species (ROS) production. As possible sources of oxidative stress, NADPH oxidases (NOX) are centrally involved in the control of the malignant state: activated NOX enzymes use NADPH as an electron donor to reduce molecular oxygen generating superoxide anion radical (O2•−), which can act extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. Excessive superoxide can be dismutated to H2O2. by superoxide dismutases (SOD). Objectives: To investigate whether chemical inhibition of NOX or removal of extracellular superoxide by exogenous SOD influences tumor cell survival depending on p53 status or metabolic stress. Experimental: Genetically-matched human melanoma cells with unequal p53 mutational status were exposed to glucose or serum starvation under normoxia, physical hypoxia and/or chemical hypoxia .Differential cell survival was assesed by live-dead fluorometric assays or crystal violet staining. Mechanisms influencing response to glucose limitation were studied by specific immune blotting. Results: Lower cell proliferation under normoxia in 1 % serum was attenuated by chemically or enzymatically decreasing superoxide or by exposing them to hypoxia. Diphenylene iodonium preferentially restored the metabolic activity of mutant p53 cells in 1 % serum, whereas SOD acted preferentially on wt p53 cells under comparable conditions.

Apoptosis-associated PARP cleavage under normoxia in 2.5 mM glucose and 5 % dialyzed serum was counteracted by SOD only in wt p53 cells. Cell death induced by CoCl2 preconditioning in the absence of physical hypoxia was preferential for mut p53 C8161 melanoma and reversed by SOD. Conclusions: Our results support a cytotoxic role for Nox-mediated free radical production in cancer cell death under metabolic stress, and suggests that p53 mutation increases superoxide-mediated toxicity during limitation of intracellular glucose and/ serum-derived growth factors.

Citation Format: Valery Chavez-Perez, Mary Strasberg-Rieber, Manuel Rieber. Superoxide modulates tumor cell survival in response to hypoxia and metabolic stress: role of p53 status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1890. doi:10.1158/1538-7445.AM2013-1890
Abstract 1896: Bioluminescent assays for investigating cell metabolism.
Authors: Leippe Donna , Sobol Mary , Duellman Sarah , Vidugiriene Jolanta , Zhou Wenhui , Vidugiris Gediminas , Good Troy , Bernad Laurent , Meisenheimer Poncho , Cali James
Published: August 2014

Cell metabolism is an important and expanding area of research in the field of cancer biology. Simple, rapid assays for studying cellular metabolic pathways, their enzymes, metabolites and cofactors can facilitate these studies. We have developed two novel bioluminescent technologies, one for monitoring cell viability, and the other for monitoring changes in cellular nicotinamide adenine dinucleotides, that can facilitate the study of cell metabolism and its regulation.

The bioluminescent cell viability assay is based on the metabolic activity of live cells. Metabolically active cells reduce a proluminogenic substrate in vivo which is then detected by a luciferase enzyme in the media. The luminescence output is correlated with the number of viable cells. The assay is non-toxic, sensitive (detects less than 10 cells/well) and has a large dynamic range (S/B>100). The key, differentiating feature of this assay is the incorporation of the recently developed stable and very bright NanoLuc™ luciferase and its profurimazine substrate, which allows changes in viability to be monitored in real-time and continuously, over extended periods of time, from the same sample.

Three homogeneous, one-step, bioluminescent assays have been developed for the detection of nicotinamide adenine dinucleotides, specific for: (1) reduced forms NADH and NADPH, (2) non-phosphorylated forms NAD and NADH, and (3) phosphorylated forms NADP and NADPH. The assays are based on the reaction of the enzyme diaphorase with a proluciferin substrate, which in the presence of NADH or NADPH is converted to luciferin, a substrate for luciferase. These assays are sensitive (LOD ≤ 50 nM) and have large maximum signal windows (S/B > 100), two advantages over currently available colorimetric and fluorescent detection methods. They are also robust, and amenable to automated and high-throughput protocols (Z’ > 0.8). The high sensitivity, one-step reagent addition, and no requirement for sample processing, permit an in-well protocol for the assay of cells directly in culture plate wells. Changes in cellular NAD levels can be rapidly monitored, as demonstrated using FK866, an inhibitor of NAD biosynthesis. FK866 caused a decrease in NAD levels in multiple cell types with good signal windows (S/B > 10) and expected pharmacology (IC50 values = 1-10 nM).

Citation Format: Donna Leippe, Mary Sobol, Sarah Duellman, Jolanta Vidugiriene, Wenhui Zhou, Gediminas Vidugiris, Troy Good, Laurent Bernad, Poncho Meisenheimer, James Cali. Bioluminescent assays for investigating cell metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1896. doi:10.1158/1538-7445.AM2013-1896