bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5341: Clinical relevance of metastasis-related miRNAs in early breast cancer.
Authors: Markou Athina N. , Yousef George M. , Liang Yu , Stathopoulos E , Georgoulias Vassilios , Lianidou Evi S.
Published: August 2014

Introduction

It was recently demonstrated that miRNAs control breast cancer metastasis by influencing multiple metastatic steps. In this study we firstly evaluated the clinical relevance of specific miRNAs -recently shown to correlate with metastasis in breast cancer by exploring their expression profile in FFPEs samples of early breast cancer patients in respect to the clinical outcome of the patients. Finally, we investigated the correlations between the expression of these selected miRNAs and other parameters such as ER and PR expression.

Materials and Methods

One hundred and twelve (112) FFPE blocks were obtained from breast tumors of early breast cancer patients (follow-up period of 11 years) and 13 mammoplasty specimens from healthy tissues which were used as controls. The median overall survival (OS) and disease free interval (DFI) were 84 and 68 months respectively. We have chosen to quantify the expression of six metastasis related miRNAs, namely: miR-10b, miR-2,1 miR-205, miR-210, miR-335 and let-7a by RT-qPCR in the LightCycler (Roche). HER2, ER and PR expression of the primary tumors was detected by immunohistochemistry with the monoclonal antibody CB11 and with monoclonal antibodies to ER and PR, respectively, using the OPTIMAX automated system. For all miRNAs relative expression was determined using the ΔCq approach and expression values were normalized to miR-191 which has been shown to be a suitable reference miRNA.

Results

Kaplan-Meier survival curves demonstrated that patients with high miR-21 expression (n=56) had a significant shorter DFI than those with low miR-21 expression (P=0.043). Moreover, patients with low expression of miR-205 (n=57) had both shorter DFI and OS time than those with high expression levels (P=0.040 and P=0.047 respectively). However, the expression levels of the other four miRNAs were not related with the DFI and OS. We also found that in the ER-negative subgroup (n=46), disease relapse was more common in patients where miR-10b was over-expressed (P=0.019), while in the PR-negative subgroup (n=47), disease relapse was more common in patients with miR-21 overexpression (P<0.001) or miR-10b overexpression (P =0.016). Finally, in the group of PR-negative patients we noticed that patients who overexpressed miR-21 had shorter OS (P=0.021).

Conclusions

Quantification of metastasis related miRNAs expression in primary tumors can give important prognostic information in patients with early breast cancer.

Citation Format: Athina N. Markou, George M. Yousef, Yu Liang, E Stathopoulos, Vassilios Georgoulias, Evi S. Lianidou. Clinical relevance of metastasis-related miRNAs in early breast 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 5341. doi:10.1158/1538-7445.AM2013-5341
Abstract 5347: MiRNA expression profile of tumor microenvironment in murine liver metastatic model.
Authors: Marquez Joana , Schaub Arianne , Unzurrunzaga Maite , Garcia Patricia , Badiola Iker
Published: August 2014

Colorectal cancer (CRC) is the second most recurrent cancer type and one of the most lethal diseases around the worldwide. The lethality of the CRC is due to the metastasis and the principal target is the liver. Although the main responsible of the metastasis is the cancer cell we can not obviate the crucial role of the host organ and how the cells of the host organ support the progression of the disease. The crosstalk of the tumor microenvironment with the tumor has been deeply studied from the cellular point of view but the regulatory pathways of these processes still remain unknown. The epigenetic regulation has been postulated as one important process implicated in the regulation of cellular changes. The microRNA (miRNA) expression is one of the variants of the epigenetic regulation. In this work we study the deregulation of the miRNA expression in the metastasized liver respect to healthy condition using a murine in vivo liver metastasis model.

We inoculated CT26 murine colon carcinoma cell intrasplenically in BALB/c mice and after 15 days we sacrificed the animals to obtain four liver cell types (hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells). Then we isolated the total RNA and performed gene expression and miRNA expression microarrays (Agilent) comparing the tumor received cells with control cells (PBS injected animal's cells). The gene expression microarray helps us finding the biological and cellular sense of the deregulated miRNAs and could be an important data source to elucidate the therapeutic option of each deregulated miRNAs. An in vivo approach is a more realistic model that can show more elements implicated in the crosstalk of the different liver cell types. This fact show us several results in the different cell types with expected and unexpected new miRNA deregulations. For instance downregulation of miR-16 and miR-15b and upregulation of miR-138 has been found in hepatic stellate cells with important role in activation proliferation and apoptosis of this cell type and we also observed deregulation of miR-135 in liver sinusoidal cells. These are some of the examples of a large amount of miRNA identified. Nowadays we are checking the role of each deregulated miRNA in the cellular processes to verify their implication in the metastatic progression and postulate them as therapeutic targets.

Citation Format: Joana Marquez, Arianne Schaub, Maite Unzurrunzaga, Patricia Garcia, Iker Badiola. MiRNA expression profile of tumor microenvironment in murine liver metastatic model. [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 5347. doi:10.1158/1538-7445.AM2013-5347
Abstract 5369: Methylation profile of markers for epithelial-mesenchymal transition and stem cell in tamoxifen-resistant MCF-7 breast cancer cell line.
Authors: Lee Youngseok , Kim Aeree , Kim Han-Kyeom
Published: August 2014

DNA methylation plays a key role in the epigenetic-based regulation of gene expression. Several genes that showed increased hypomethylations of gene promotors in antiestrogen-resistant breast cancer cells were found to be increased in tumor cells and possess oncogenic activity. The purpose of this study is to investigate the differences of methylation profiles, especially EMT markers, between tamoxifen-sensitive breast cancer cell lines and tamoxifen-resistant cell lines using MCF7 and tamoxifen-resistant MCF7 (MCF7-R) cell lines, and then to indentify the presence of the identified markers in the breast tissues of recurred patients with antiestrogen treatments. Bisulphite-converted genomic DNA was assayed on Infinium Human Methylation 450K Beadchips using the Illumina Infinium HD methylation assay kit using four groups of the cells including MCF7, MCF7-R, MCF+E2 and MCF7-R+E2. . In MCF7-R cells, 31023 CpG sites showed altered methylations, with 13617 hypomethylated and 17406 hypermethylated, compared with MCF7. The numbers of hypomethylated and hypermethylated CpG sites in the promoter regions were 4719 and 3347, respectively. Among the well-known EMT markers, the genes meeting the results of methylation status selected were CTNNB1, SNAIL2, TWIST1, LEF1, GSC and ETS1. Although the results of the current study suggest that aberrant DNA methylations of EMT markers are not the main force driving the molecular biology of resistance against antiestrogen therapy, these will contribute and result in to some extent.

Citation Format: Youngseok Lee, Aeree Kim, Han-Kyeom Kim. Methylation profile of markers for epithelial-mesenchymal transition and stem cell in tamoxifen-resistant MCF-7 breast cancer cell line. [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 5369. doi:10.1158/1538-7445.AM2013-5369
Abstract 5372: Cocaine enhances HIV-1 integration by modulating the epigenetic signatures of CD4+ T cells.
Authors: Addai Amma , Dash Chandravanu
Published: August 2014

Human Immunodeficiency Virus (HIV-1) has claimed the lives of over 25 million people. Illicit drug use serves as a co-factor for HIV/AIDS. Cocaine, a commonly used drug among HIV-1 positive individuals, serves as a cofactor for HIV-1 infection and progression to acquired immunodeficiency syndrome (AIDS). Previous studies suggest that cocaine increases HIV-1 replication in cell cultures, peripheral blood mononuclear cells (PBMCs) and animal models. However, the mechanism by which cocaine enhances HIV-1 replication in CD4+ T cells remains largely unknown. During HIV-1 replication, the viral genome is reverse transcribed into a double stranded DNA (dsDNA) by HIV-1 reverse transcriptase (RT). This viral dsDNA is transported into the nucleus in the form of a pre-integration complex (PICs) that contains the viral integrase (IN) and other viral and cellular proteins. Integration is a critical step in the HIV-1 life cycle. The main objective of this study is to examine the effects of cocaine on HIV-1 integration by its modulation of epigenetic signatures of CD4+ T cells. However, there are no studies on cocaine-induced integration in CD4+ T cells which serves as the main targets for HIV-1 replication in vivo. A correlation between DNA methylation and HIV-1 integration is yet to be demonstrated biochemically and mechanistic information is limited. Experimental strategy for in vitro study involves the production of infectious HIV-1 NL4.3 virus, isolation of HIV-1 PICs from SupT1 lymphoma infected cells and carrying out integration assay. Our preliminary data demonstrates cocaine and DNA demethylating agent (5-aza-2′-deoxycytidine) both enhance HIV-1 integration. The isolated PICs are active in integrating viral DNA into target DNA and integration was more efficient in unmethylated DNA than methylated DNA. To confirm integration activity, we performed quantitative nested PCR analysis using primers specific to the target and viral DNA. We propose that results from this project will help us better understand the role of cocaine in HIV-integration by modulating the epigenetic signatures of CD4+ T cells. Furthermore, understanding the molecular details of HIV-1 integration will aid in the discovery of novel targets that can synergize with integrase inhibitors as part of an approach to cure HIV/AIDS.

*Amma Addai is supported by T32 grant #2T32H007735-17 from

NIH/NHLBI to Dr. S. E. Adunyah

Citation Format: Amma Addai, Chandravanu Dash. Cocaine enhances HIV-1 integration by modulating the epigenetic signatures of CD4+ T 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 5372. doi:10.1158/1538-7445.AM2013-5372
Abstract 5380: Detection of OvCa1 in human malignancies.
Authors: Jackson Jessica L. , Grothaus Kristen A. , Temm Connie J. , Mayo Lindsey D. , Sanduksy George E.
Published: August 2014

Immunohistochemistry biomarkers are currently being developed to target specific proteins found in cancer cells, which could contribute to a better understanding of specific types of cancers and provide information to develop better treatments for cancer patients. The biomarker and putative tumor suppressor, OvCa1, has a function that is not well characterized. Due to a lack of staining reagents, we developed monoclonal antibodies of OvCa1 to examine multiple human malignancies. The goal of this project was to gain a better understanding of OvCa1 and its role in cancer. Primary cancers with different histological grades as well as metastatic lesions were examined with the monoclonal antibodies. Ovarian cancer tissue samples from the Indiana University Simon Cancer Center Tissue Bank were used for this study. The samples were fixed in neutral buffered formalin and processed into paraffin blocks. The tissues were cut using a microtome and placed on slides. Staining of the OvCa1 antibody was completed using immunohistochemistry (IHC) techniques. Thirty-one samples were evaluated including low grade tumors, medium grade tumors, high grade tumors, and metastatic ovarian carcinomas. All cases revealed a range of staining intensity with the OvCa1 antibody. The results indicated that OvCa1 for the high grade, stage 3/4, ovarian carcinomas had the highest staining intensity. Medium grade tumors had less OvCa1 expression. The metastatic tumors had less staining than any of the other three grades. Immunostaining was observed primarily in the cytoplasm and nucleus of the tumor cells. In addition, we evaluated approximately 20 tumors from various different organs. These included prostate, breast, spleen, lung, colon, stomach, and kidney tumors, which were positive for immunostaining with the OvCa1 antibody. In summary, the results indicated that all histological grades expressed the biomarker, OvCa1, and the staining intensity was highest in the high grade, stage 3/4, tumors. Our preliminary studies demonstrated a further need to delineate OvCa1 as a potential biomarker, which could be used for early detection and diagnosis of ovarian cancer.

Citation Format: Jessica L. Jackson, Kristen A. Grothaus, Connie J. Temm, Lindsey D. Mayo, George E. Sanduksy. Detection of OvCa1 in human malignancies. [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 5380. doi:10.1158/1538-7445.AM2013-5380
Abstract 5385: Gene expression changes downstream of APC loss predict tumor phenotype.
Authors: Pehar Andjela , Cole Jennifer M. , Prosperi Jenifer R.
Published: August 2014

The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in up to 70% of human breast cancer cases. Our previous studies demonstrated that Apc mutation advances the MMTV-Polyoma Middle T Antigen (PyMT) model of breast cancer, with an increase in signaling downstream of focal adhesion kinase (FAK)/Src/JNK activation in MMTV-PyMT;ApcMin/+ tumors. In addition, the majority of tumors that arose in the MMTV-PyMT;ApcMin/+ animals were classified as adenosquamous carcinoma as compared to tumors from MMTV-PyMT;Apc+/+ animals, which were solid carcinomas. Given the heterogeneous nature of these tumors, laser capture microdissection (LCM) of the adenosquamous carcinoma in MMTV-PyMT;ApcMin/+ tissues followed by quantitative real-time RT-PCR was performed to identify gene expression changes responsible for the altered tumor phenotype. In parallel studies, gene expression microarrays were performed using triplicate samples of the MMTV-PyMT;Apc+/+ and MMTV-PyMT;ApcMin/+ cell lines. RNA samples were subjected submitted to quality control via the Agilent Bioanalyzer and were hybridized to Affymetrix Mouse Genome 430 2.0 microarrays. Data analysis was performed using the Gene-Go software, and significant changes were confirmed using qRT-PCR. Multiple changes consistent with the phenotypic and proliferative changes downstream of APC loss were identified, including changes in expression of cyclooxygenase-2 (COX-2). The findings presented here indicate that APC-mediated gene expression changes can be used to predict tumor phenotype and downstream therapeutic targets.

Citation Format: Andjela Pehar, Jennifer M. Cole, Jenifer R. Prosperi. Gene expression changes downstream of APC loss predict tumor phenotype. [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 5385. doi:10.1158/1538-7445.AM2013-5385

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5387: Integrative analysis of transcriptomic and metabolomic data reveals a critical role for aminosugar metabolism in prostate cancer.
Authors: Panzitt Katrin , Shojaie Ali , Putluri Nagireddy , Basu Sumanta , Putluri Vasanta , Samanta Susmita , Ittmann Michael , Vergara Ismael , Michailidis George , Palapattu Ganesh , Sreekumar Arun
Published: August 2014

Prostate Cancer is the second most common cause of cancer-related death in men in the US. Like other tumors, prostate cancer development and progression is dictated by multiple molecular events that include changes in levels of genes, transcripts, proteins and metabolites. To better understand the biology of prostate cancer development it is essential to integrate these disparate yet related datasets. Our laboratory has identified matched transcriptomic, proteomic and metabolomic changes in localized prostate cancer relative to adjacent benign tissue as well in metastatic disease compared to organ-confined tumor. To study this using a System's Biology approach we have recently embarked on a pilot study aimed at integrating transcriptomic and metabolomic data to obtain biochemical pathways that key to prostate cancer development. Using an in-house network-based enrichment strategy, amino sugar metabolism was found to be significantly enriched in organ-confined prostate cancer but not in metastatic disease. Amino sugar metabolism describes the utilization of glucose-derived carbon and amino acid (mostly glutamine)-derive nitrogen to produce glucosamines. These amino sugars participate in synthesis of immune modulatory compounds as well as in glycosylation cascades. In this study, we describe the molecular analyses of Glucosamine-6 phosphate-N-acetyl Transferase (GNPNAT1), a key enzyme that converts D-glucosamine 6-phosphate to N-acetyl-D-glucosamine 6-phosphate, in prostate cancer. Our results indicate upregulation of GNPNAT1 in organ-confined prostate cancer as compared to benign adjacent tissue as well as metastatic tissue and regulation of the pathway by androgen. Stable knockdown of GNPNAT1 in androgen dependent LNCap cells leads to diminished cell growth and cell cycle arrest. In contrast, growth and cell cycle are not affected by the knockdown of GNPNAT1 in androgen independent C4-2 cells. Knockdown of GNPNAT1 in C4-2 cells enhances invasiveness which is not observed in LNCap knockdown cells. In this work we show that GNPNAT1 is linked to androgen receptor (AR) action and thus is a critical enzyme for the survival of androgen dependent prostate cancer cells.

Citation Format: Katrin Panzitt, Ali Shojaie, Nagireddy Putluri, Sumanta Basu, Vasanta Putluri, Susmita Samanta, Michael Ittmann, Ismael Vergara, George Michailidis, Ganesh Palapattu, Arun Sreekumar. Integrative analysis of transcriptomic and metabolomic data reveals a critical role for aminosugar metabolism in prostate 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 5387. doi:10.1158/1538-7445.AM2013-5387
Abstract 540: Role of EphB4-EphrinB2 as novel target in Her2 positive breast cancer.
Authors: Duarte A. , Quaresma J. , Trindade A. , Tripathy P. , Liu X. , Krasnoperov V. , Liu R. , Gill P.S.
Published: August 2014

Background: Her2 amplification drives tumor growth in 20% of breast cancers. Her2 targeted therapies have improved survival, but eventual failure occurs and additional targeted therapies to overcome resistance are needed. EphB4 and EphrinB2 receptor kinase -ligand pair is induced in Her2 positive tumors and is a novel target.

Methods: Her2 positive tumor cell lines and Her2 transgenic mouse lines were studied for expression of EphB4-EphrinB2. The role of EphB4-EphrinB2 was examined by knock down with shRNA. In vivo tumor xenograft studies with Her2 positive cell lines resistant or sensitive to Her2 targeted therapy, and Her2 transgenic mouse were treated with soluble decoy receptor (soluble EphB4-Albumin fusion protein). Tumor response and activation status of Her2 were examined.

Results: EphB4 and EphrinB2 are induced in Her2 positive tumor cell lines with further induction in trastuzumab (Herceptin) resistant tumor lines. Knock down of EphB4 with shRNA reduced the cell viability by nearly 80% compared to control shRNA. In vivo studies with sEphB4-Albumin also reduced the tumor growth by 70-80% accompanied by the reduction in pHer2 and pEGFR. No effect was seen on pHer3. Combination with lapatinib and sEphB4-Albumin was additive. sEphB4-Albumin inhibited tumor growth in Her2 resistant tumor cell lines by 56%.

Conclusion: EphB4 and EphrinB2 are induced by Herceptin sensitive and resistant Her2 positive tumors. EphB4 provides survival advantage to Her2 positive trastuzumab sensitive and resistant tumors. sEphB4-Albumin inhibits tumor growth as single agent in both trastuzumab sensitive and resistant tumors. sEphB4-Albumin is currently in the clinical development and thus worthy of investigation in Her2 positive cancers.

Citation Format: A. Duarte, J. Quaresma, A. Trindade, P. Tripathy, X. Liu, V. Krasnoperov, R. Liu, P.S. Gill. Role of EphB4-EphrinB2 as novel target in Her2 positive breast 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 540. doi:10.1158/1538-7445.AM2013-540
Abstract 5407: The novel roles of BRCA1/ATM-associated BRAT1 in glucose/mitochondrial metabolism and tumor cell growth.
Authors: So Eui Young , Ouchi Toru
Published: August 2014

BRAT1 (BRCA1-associated ATM activator) was identified as a novel DNA-damage response protein. It physically interacts with both BRCA1 and ATM, and is required for ATM-activation under conditions of cell stress. We have recently discovered that it also binds to DNA-PKs, implicating the broad role of BRAT1 in DNA repair as well as in DNA damage response. By taking advantage of BRAT1 knockdown HeLa and U2OS cells, we found that loss of BRAT1 expression significantly decreased cell proliferation and tumorigenecity both in vitro and in vivo. Cell motility is remarkably lowered when BRAT1 is depleted. These results demonstrate novel function of BRAT1 in cell proliferation, migration, and tumorigenesis of cancer cells. Interestingly, glucose uptake and production of mitochondrial ROS (reactive oxygen species) are highly increased in BRAT1 knockdown. These findings suggest novel functions of BRAT1 in regulating glucose metabolism and mitochondrial function. Functional interaction of BRCA1/ATM pathway and glucose/mitochondria metabolism are discussed.

Citation Format: Eui Young So, Toru Ouchi. The novel roles of BRCA1/ATM-associated BRAT1 in glucose/mitochondrial metabolism and tumor cell growth. [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 5407. doi:10.1158/1538-7445.AM2013-5407
Abstract 5409: Cetuximab downregulates lactate dehydrogenase-A, leading to inhibition of glycolysis and reversal of the Warburg effect in HNSCC cells.
Authors: Lu Haiquan , Li Xinqun , Fan Zhen
Published: August 2014

Cetuximab is an anti-human epidermal growth factor receptor (EGFR) monoclonal antibody currently approved for treatment of patients with head and neck squamous cell carcinoma (HNSCC) in combination with conventional radiotherapy and chemotherapy. Previous studies showed that cetuximab downregulates the alpha subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1α) through inhibition of EGFR-mediated downstream cell signaling and that downregulation of HIF-1α is required for cetuximab-induced antiproliferative effects; however, the underlying mechanism has not been established. In this study, we explored the mechanism from the perspective of cancer metabolism. HIF-1 plays a critical role in reprogramming cancer metabolism towards aerobic glycolysis (i.e., the Warburg effect), which is critically important for supplying cancer cells with the biomass needed for cell proliferation. We found that cetuximab downregulated lactate dehydrogenase A (LDH-A) and inhibited glycolysis in cetuximab-sensitive cells in a HIF-1α downregulation-dependent manner. HNSCC cells with acquired resistance to cetuximab expressed a high level of HIF-1α and were highly glycolytic. Overexpression of a HIF-1α mutant conferred resistance to cetuximab-induced G1-phase cell cycle arrest, which could be overcome by knockdown of LDH-A expression. Addition of a small molecule LDH-A inhibitor markedly enhanced the response of cetuximab-resistant cells to cetuximab. Cetuximab had no noticeable inhibitory effect on glycolysis in nontransformed cells, in which glycolysis is followed by oxidative phosphorylation in the mitochondria. These findings provide novel mechanistic insights into cetuximab-induced antitumor effects from the perspective of cancer metabolism and suggest novel strategies for enhancing the response of cancer patients to cetuximab therapy.

Citation Format: Haiquan Lu, Xinqun Li, Zhen Fan. Cetuximab downregulates lactate dehydrogenase-A, leading to inhibition of glycolysis and reversal of the Warburg effect in HNSCC 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 5409. doi:10.1158/1538-7445.AM2013-5409
Abstract 5421: Sensitivity to dichloroacetate is determined by PDK expression.
Authors: Gang Bevan P. , Rooke Melissa , Mishra Sheenu , Dahlstrom Jane E. , Blackburn Anneke C. ,   Cancer Metabolism and Genetics Group.
Published: August 2014

The Warburg effect occurs in 90% of tumours, where there is a high rate of glycolysis even in the presence of oxygen. This results in increased lactate production and reduced pyruvate oxidation. Dichloroacetate (DCA) is used clinically for congenital lactic acidosis and can reverse the Warburg effect by inhibiting the pyruvate dehydrogenase kinases (PDKs), activating pyruvate dehydrogenase thus promoting oxidative metabolism of pyruvate. The PDKs have different sensitivities towards DCA inhibition (PDK2>PDK4>PDK1>>PDK3). We have investigated the effects of DCA on cancer growth in vivo and in vitro, and examined the expression of the PDK isoforms to explain variation of cancer cell responses to DCA.

DCA halted the growth of established V14 mouse mammary tumours in vivo, but was ineffective against 4T1 mouse mammary tumours. In 8 human cancer cell lines of breast, colon, pancreatic and prostate origin, 5 mM DCA (48 hr) inhibited growth by 5-40% in vitro. Western blotting for expression of PDK isoforms revealed that the most sensitive T-47D cells expressed PDK2 and low levels of the other PDKs, whereas less sensitive cells expressed high levels of PDK1 and/or PDK3. In siRNA knockdown (kd) experiments, PDK3-kd in MCF7 cells (low DCA sensitivity, high PDK3 levels) increased sensitivity to DCA by 30%, while DCA did not further inhibit growth of PDK2-kd T-47D cells, confirming that sensitivity to DCA growth inhibition is determined by the PDK profiles. PDK1 & 3 are up-regulated in hypoxia, which may reduce the effectiveness of DCA. To the contrary, DCA increased apoptosis in hypoxia in MCF7 cells, suggesting that DCA can enhance effects of other stressors/drugs, an area of ongoing investigation.

Use of the PDK profiles to target DCA sensitive tumours will improve the outcomes of clinical trials using DCA that are currently underway

Citation Format: Bevan P. Gang, Melissa Rooke, Sheenu Mishra, Jane E. Dahlstrom, Anneke C. Blackburn, Cancer Metabolism and Genetics Group. Sensitivity to dichloroacetate is determined by PDK expression. [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 5421. doi:10.1158/1538-7445.AM2013-5421

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5426: Severe hypoxia drives the increased glucose demand in ascites carcinomas.
Authors: Li Xiao-Feng
Published: August 2014

Warburg reported his discovery in Ehrlich ascites carcinoma model, that in the presence of ample oxygen (21% or 150 mmHg O2), cancer cells utilize glucose by “aerobic glycolysis” (Warburg effect), apparently, ascites carcinoma had been assumed to be oxic. However, during past decade, growing evidences indicated that hypoxic cancer cells had evaluated radiolabeled glucose analog18F-FDG uptake than oxic cancer cell in vitro and in vivo. The objective of this study was to observe the oxygen status of ascites fluid and ascites carcinoma in mouse model of ascites carcinoma and its effect on glucose metabolism. Methods: Ascites carcinoma (floating single cells or clusters) and serosa tumors (attached to serosa) were induced by HT29, A549 or MDA-MB-231 cancer cells in the mouse peritoneal cavity. Ascites pO2 was measured using OxyLite systems in ketamine/xylazene anesthetized mice. Hypoxia status in ascites carcinoma and serosa tumors was tested by immunohistochemical visualization of exogenous hypoxia marker pimonidazole binding and HIF1alpha induced glucose transporter-1 expression. Glucose metabolism of was examine by 18F-FDG autoradiography. Effect of carbogen breathing (95% O2 & 5% CO2) on 18F-FDG uptake was investigated. Results: The pO2 of ascites was measured as low as 0.90mmHg (or 0.5% O2) in mice with HT29 ascites carcinomas. In all cell lines we tested, ascites carcinomas was evident 4-6 wk after cell inoculation, all ascites tumors were stained positive for pimonidazole and glucose transporter-1 (GLUT-1), indicating severe hypoxia. Hypoxia status in serosa tumors was heterogeneous and dependent on tumor size. 18F-FDG uptake was significantly higher in ascites and sub-millimeter serosa tumors than in larger ones (generally greater than 1 mm), the former was stained positive for pimonidazole and GLUT-1, but the latter was not. Carbogen breathing (2hrs) significantly reduced 18F-FDG uptake and pimonidazole binding in ascites carcinoma and submillimeter serosa tumors. GLUT1 expression was not affected. Conclusions: In mouse model of ascites carcinoma, ascites and floating ascites carcinoma are severely hypoxic which is in contradict with ample oxygen condition of Ehrlich ascites carcinoma model where “Warburg effect” derived from. Glucose utilization measured by 18F-FDG uptake is oxygen dependent in both ascites carcinoma and serosa tumors, this is unexplained sole by “aerobic glycolysis” of glucose metabolism, thus challenging Warburg effect theory.

This study was supported by Kentucky Lung Cancer Research Program Award

Citation Format: Xiao-Feng Li. Severe hypoxia drives the increased glucose demand in ascites carcinomas. [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 5426. doi:10.1158/1538-7445.AM2013-5426
Abstract 5431: PPAR alpha independent effect of fenofibrate on glioblastoma cancer metabolism.
Authors: Wilk Anna M. , Zapata Adriana M. , Mullinax Jennifer R. , Wyczechowska Dorota D. , Reiss Krzysztof
Published: August 2014

Glioblastoma multiforme is the most common and aggressive primary brain tumor in adults. It is characterized by a rapid cell growth, CNS infiltration and is resistant to all known anticancer regimens. Therefore, development of effective therapies against these malignant brain tumors represents challenge for contemporary medicine. Anti-neoplastic potential of a common lipid-lowering drug, fenofibrate, is gaining a lot of attention, however, the precise mechanism of fenofibrate anti-cancer action remain to be elucidated. Fenofibric acid, which is an active metabolite of fenofibrate, is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα), which transcriptional activity is expected to shift energy metabolism from glycolysis to fatty acid β-oxidation, therefore, it may be toxic to glycolytic tumor cells (Warburg effect). In parallel, to PPARα- dependent action, we have observed a strong PPARα- independent effect of fenofibrate on cancer cell energy metabolism, which was specific only to fenofibrate but not to fenofibric acid or to other PPARα agonists. We detected fenofibrate in mitochondrial membrane fractions of LN-229 cells treated with fenofibrate by using HPLC and subcellular fractionation. The accumulation of fenofibrate in mitochondrial membrane was accompanied by significant change in mitochondrial function, including loss of mitochondrial membrane potential, dramatic loss of oxygen consumption and lack of respiratory capacity measured as oxygen consumption rate (OCR). At the same time, fenofibrate increased the level of glycolysis (extracellular acidification rate, ECAR), possibly as compensation for impaired oxidative phosphorylation. In addition, fenofibrate-induced reduction of ATP levels triggered phosphorylation of AMPK and activation of autophagy.

In conclusion, our results demonstrate two seemingly independent mechanisms of fenofibrate anticancer action: PPARα-dependent, which shifts cell metabolism from glycolysis to fatty acid β-oxidation and PPARα-independent, which causes accumulation of fenofibrate in cell membranes and inhibition of oxidative phosphorylation. Both mechanisms counteract to make this drug very effective against Glioblastoma tumors causing inhibition of cell growth, survival and finally leading to massive apoptosis of these practically incurable tumors.

Citation Format: Anna M. Wilk, Adriana M. Zapata, Jennifer R. Mullinax, Dorota D. Wyczechowska, Krzysztof Reiss. PPAR alpha independent effect of fenofibrate on glioblastoma cancer 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 5431. doi:10.1158/1538-7445.AM2013-5431

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5433: Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity in ovarian cancer cells.
Authors: Zhang Shu , Mao Weiqun , Sood Anil K. , Jennings Nicholas B. , Rodriguez-Aguayo Cristian , Lopez-Berestein Gabriel , Li Zong-Fang , Bast Robert C. , Le Xiao-Feng
Published: August 2014

Knockdown of certain glycolytic enzymes can affect ovarian cancer cell growth and enhance sensitivity to paclitaxel. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) is the cardiac isoform of the most important glycolytic regulator, phosphofructokinase-2, From the TCGA database of >300 ovarian cancers, the PFKFB2 gene ranked in the top 33% of overexpressed genes and in the top 19% of genes with copy number gain. We found that PFKFB2 mRNA was upregulated in 10 of 12 ovarian cancer cell lines when expression of the gene was compared to that in normal ovarian epithelial cells in culture. Knockdown of PFKFB2 with siRNA markedly inhibited cell proliferation and increased paclitaxel sensitivity in p53 wild type ovarian cancer cell lines as demonstrated in short-term cytotoxicity and long-term clonogenic assays. Liposome encapsulated PFKFB2 siRNA significantly inhibited growth (P<0.05) of human ovarian cancer xenografts in two ovarian cancer models (Hey A8 and A2780); even greater growth inhibition was observed when PFKFB2 siRNA treatment was combined with paclitaxel. Cell growth inhibition in cell culture was linked to G1 arrest and to a modest increase in apoptosis. Induction of p21Cip1 and p27Kip1 protein by PFKFB2 knockdown contributed to the G1 arrest, since silencing p21Cip1 and p27Kip1 dramatically reduced PFKFB2 knockdown-induced G1 arrest. PFKFB2 knockdown also modulated the expression of CDK2 and the phosphorylation of p27Kip1. Induction of p53 protein contributed to both PFKFB2 knockdown-induced G1 arrest and to apoptosis, since silencing p53 markedly alleviated PFKFB2 knockdown-induced G1 arrest and apoptosis. PFKFB2 knockdown inhibited expression of the ABC transporter family members ABCC4 and ABCG2 at the level of transcription, possibly contributing to enhanced paclitaxel retention and increased cytotoxicity after PFKFB2 knockdown. Taken together, these results suggest that PFKFB2 protein may regulate cell growth and sensitivity to paclitaxel by multiple mechanisms, particularly in p53 wild type ovarian cancers. As methods to deliver siRNA evolve in the clinic, PFKFB2 may provide a novel target for enhancing sensitivity to paclitaxel-based chemotherapy in patients with low grade serous ovarian cancer where p53 function remains intact.

Citation Format: Shu Zhang, Weiqun Mao, Anil K. Sood, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Zong-Fang Li, Robert C. Bast, Xiao-Feng Le. Knockdown of the glycolytic enzyme PFKFB2 induces growth inhibition and enhances paclitaxel sensitivity 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 5433. doi:10.1158/1538-7445.AM2013-5433
Abstract 5436: Oct4 interaction with Hmgb2 regulates Akt signaling and the pluripotent gene expression signature.
Authors: Campbell Pearl A. , Stewart David J. , Rudnicki Michael A.
Published: August 2014

Akt signaling impacts multiple cellular functions associated with the hallmark characteristics of pluripotency such as cell cycle progression, cell growth, and enhanced cellular survival. Constitutive Akt activation promotes the pluripotent state and has also been implicated in several forms of cancer. Recent studies have suggested that while Akt activation may be a positive factor, the active nuclear form of Akt is associated with poor prognosis for many cancers, including lung cancer. Here, we describe a novel transcriptional regulatory feedback loop between Oct4, Hmgb2, and Akt. We show that Akt signaling promotes Oct4 phosphorylation and stability. This maintains interaction with Akt, Hmbg2 and other members of the SET complex such as Nme1 and Set. Upon differentiation, when Akt is inactive, their decreased interaction is observed. A functional genomics approach was therefore employed to determine the relevance of Oct4 interaction with Hmgb2 in maintaining the pluripotent state. Despite its previously described role in Polycomb group recruitment and transcriptional repression in Drosophila, we find that Hmgb2 knockdown results in decreased Akt signaling and decreased expression of many Oct4 transcriptional targets including genes implicated in cell cycle regulation, DNA repair, chromatin structure, as well as the pluripotency regulators Oct4, Sox2, and Nanog. Taken together, our results suggest that Oct4, Hmgb2, and the SET complex participate in a PTM-dependent feed forward regulatory loop that promotes active Akt signaling and the pluripotent gene expression signature. Future work will be focused upon dissecting the role of this network in maintaining constitutive nuclear Akt activation and promoting the primitive epigenetic state found in cancer. These findings may provide insight into the role of Akt in lung cancer chemoresistance and suggest alternate targets for more efficacious treatment.

Citation Format: Pearl A. Campbell, David J. Stewart, Michael A. Rudnicki. Oct4 interaction with Hmgb2 regulates Akt signaling and the pluripotent gene expression signature. [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 5436. doi:10.1158/1538-7445.AM2013-5436
Abstract 5438: Gene expression profiles resulting from stable and transient loss of p53 mirrors its role in tissue differentiation.
Authors: Couture Oliver , Lombardi Eric , Davis Kendra , Hays Emily , Chandar Nalini
Published: August 2014

The tumor repressor gene, p53, is involved in a variety of cellular activities ranging from stress, differentiation and cell cycle regulation. In our previous studies we have shown p53’s transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression while the same is absent in p53 null mice. This deficiency of p53 has also been shown to produce more osteosarcomas than a complete loss of p53. This suggests that the presence of p53 is able to modify the environment within pre osteoblasts based on its ability to regulate key bone specific genes. In the present study we compared changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels in C2C12 cells capable of both myoblast and osteoblast differentiation transiently, and compared the changes in gene expression of candidate genes to cells with stable p53 knockdown. Using a PCR array to assay p53 target genes, we have found differential expression profiles when comparing stable versus transient knockdown. As expected several of genes that were profoundly affected after transient p53 loss were related to apoptosis and cell cycle regulation. Stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles suggesting that long term effect of p53 loss affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were also validated by measuring promoter activities of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression profoundly affecting normal tissue physiology than its acute loss.

Citation Format: Oliver Couture, Eric Lombardi, Kendra Davis, Emily Hays, Nalini Chandar. Gene expression profiles resulting from stable and transient loss of p53 mirrors its role in tissue differentiation. [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 5438. doi:10.1158/1538-7445.AM2013-5438
Abstract 5439: Developmental checkpoint function of Δ40p53.
Authors: Zhang Piyan , Scrable Heidi
Published: August 2014

Δ40p53, an N-terminally truncated p53 isoform, is predominantly expressed in embryonic stem cells (ESCs) and during early post-implantation embryogenesis in the mouse. We have discovered that Δ40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs. Our studies revealed this isoform of p53 inhibits retinoic acid-induced differentiation and subsequent epithelial-mesenchymal transition (EMT). As ESCs differentiate, Δ40p53 cannot activate the expression of some important developmental genes, such as p21, FGF10, DKK1, lamin A and ZO-1, coincident with decreasing Erk signaling and an enhanced Wnt/β-catenin pathway. ChIP assays suggested that p21, FGF10 and ZO-1 are inhibited at the elongation step. To distinguish between the transcription regulation functions of full-length p53 and Δ40p53, we generated stable HeLa cell lines transfected with Flag-tagged p53 and Δ40p53. qPCR and ChIP assays confirmed that Δ40p53, but not full-length p53, represses gene elongation, with a concomitant decrease in acetylation of histone H4 and H3 in the promoter region. Our studies reveal that Δ40p53, but not p53, is able to block ESC differentiation by regulation of specific gene expression at the elongation step during mouse development.

Citation Format: Piyan Zhang, Heidi Scrable. Developmental checkpoint function of Δ40p53. [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 5439. doi:10.1158/1538-7445.AM2013-5439
Abstract 5453: YK-4-279 inhibits ETS-positive prostate cancer cell metastasis in a mouse xenograft model.
Authors: Rahim Said , Justvig Sarah , Hong Sung-Hyeok , Kong Yali , Brown Milton L. , Morrissey Colm , Toretsky Jeffrey A. , Uren Aykut
Published: August 2014

Chromosomal translocations involving the ETS family of transcription factors are found in a majority of prostate cancers, including the most clinically aggressive forms. These translocations produce a chimeric gene, which fuses the promoter region of an androgen responsive gene to the coding region of ETS factors, most frequently ETV1 or ERG. Over-expression of ETS factors in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer.

We recently demonstrated that the small molecule YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here we present our findings in an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously injected with LNCaP and PC-3 xenografts. Mice were treated with YK-4-279 and its effect on primary tumor size and lung metastasis was observed. YK-4-279 treatment resulted in reduced metastasis of the tumor from primary site to lungs. YK-4-279 also affected expression of ETV1 target genes such as MMP7 and MMP13. ETS fusion-negative PC-3 xenografts were unresponsive to YK-4-279.

Our results demonstrate that YK-4-279 decreases tumor growth and inhibits metastasis in fusion-positive prostate cancer cell xenografts. Therefore, YK-4-279 may have an impact on preventing metastasis in prostate cancer patients, which is a leading cause of death.

Citation Format: Said Rahim, Sarah Justvig, Sung-Hyeok Hong, Yali Kong, Milton L. Brown, Colm Morrissey, Jeffrey A. Toretsky, Aykut Uren. YK-4-279 inhibits ETS-positive prostate cancer cell metastasis in a mouse xenograft model. [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 5453. doi:10.1158/1538-7445.AM2013-5453