bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 985: Mitochondrial glutamine fermentation enhances ATP synthesis in murine glioblastoma cells
Authors: Seyfried Thomas N.
Published: June 2012

Otto Warburg first proposed that all cancer cells arise from irreversible impairment of respiration, thus requiring glucose fermentation as a compensatory energy source for maintaining viability. Glucose fermentation with lactate production in the presence of oxygen (aerobic glucose fermentation or the Warburg effect) is now recognized as a metabolic hallmark of nearly all cancers. Besides glucose, glutamine also serves as an energy metabolite for many cancer cells. It is not clear, however, how glutamine provides energy to cancer cells. We examined the influence of glucose and glutamine on ATP synthesis and viability in cultured mouse VM-M3 cells, a model for invasive human glioblastoma. The VM-M3 cells are known to have abnormal respiration. The cells were grown for 12 hrs or 24 hrs in “serum free minimal media” containing glucose alone (25 mM), glutamine alone (4.0 mM), or a combination of both energy metabolites. Using a bioluminescent-based in vitro ATP assay, we found that ATP production and cell viability was similar in VM-M3 cells grown in media containing either glutamine or glucose alone. Lactate production was significantly lower in VM-M3 cells grown in glutamine (0.24 mM) than in the cells grown in glucose (2.34 mM), indicating that these cells produce little lactate from glutamine alone. Robust synergy was found for ATP synthesis and lactate production when the cells were grown in both glucose and glutamine. Remarkably, the synergy persisted under anoxia (95% N2, 5% CO2) indicating that OxPhos did not play a major role in the synergistic interaction of glucose and glutamine. Preliminary [C13] NMR analysis showed that the cells grown in pan-labeled glutamine produced labeled succinate, aspartate, alanine, and citrate indicating that glutamine was metabolized through the TCA cycle in these cells. We propose that the glucose/glutamine energy synergy observed in the VM-M3 cells arises from linked fermentation redox couples in the cytoplasm and mitochondria that synthesize ATP largely through non-oxidative substrate level phosphorylations and possibly from an anaerobic electron transfer-based ATP synthesis at the level of fumarate reductase. In other words, the cells appear to generate ATP through simultaneous anaerobic glucose and amino acid fermentation even in the presence of oxygen. Our results support the Warburg cancer theory and suggest that besides glucose fermentation in the cytoplasm, glutamine fermentation in the mitochondria might also serve as an alternative compensatory energy source to OxPhos deficiency. Supported by Boston College Res. Fund.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 985. doi:10.1158/1538-7445.AM2011-985
Abstract 988: Upregulation of pyruvate dehydrogenase kinase 3 mediates hypoxia-induced drug resistance in colon cancer
Authors: Lu Chun-Wun , Lin Shao-Chieh , Chien Chun-Wei , Lin Shih-Chieh , Lin Bo-Wen , Lee Jenq-Chang , Tsai Shaw-Jenq
Published: June 2012

Colorectal cancer is the second leading cause of cancer-related death in both male and female worldwide. Despite the efforts of screening and advances in treatment, 50% of patients with colon cancer will ultimately die from their disease. One major hurdle of treating colon cancer is its high recurrence due to resistance to anti-cancer therapy of hypoxic tumor, a survival advantage resulting from metabolic switch of cancer cells. The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and is associated with tumor malignancy. Previous studies demonstrated that pyruvate dehydrogenase kinase-1 (PDK1) and PDK3 play an important role in the metabolic switch of cancer cells. Herein, we assessed the expression profile of PDK1 and PDK3 in colorectal cancer. Western blotting results demonstrated that levels of PDK3 were markedly upregulated in colon cancer compared to adjacent normal tissues while levels of PDK1 were decreased in cancer cells. Immunohistochemical staining (n=307) further revealed that expression of PDK3 was positively correlated with the level of hypoxia inducible factor-1α (HIF-1α) and the severity of cancer. Level of PDK3 was markedly induced by hypoxia in a HIF-1α dependent manner, while HIF-2α had no effect on PDK3 expression in colon cancer cell lines. Aberrant expression of PDK3 mediates hypoxia-induced drug resistance as knocking-down PDK3 by siRNA under hypoxia abolished such protective effect. Taken together, these data demonstrated that PDK3 is overexpressed in colon cancer and is associated with tumor malignancy. Our current findings provide novel insights about metabolic switch in cancer cells and suggest that PDK3 can be a new and pivotal target for cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 988. doi:10.1158/1538-7445.AM2011-988
Abstract 990: Metabolic shift in cancer by re-programming of mitochondrial machinery: Novel insight into Warburg-dependent mechanism
Authors: Narain Niven R. , Sarangarajan Rangaprasad , McCook John P. , Jimenez Joaquin J.
Published: June 2012

Otto Warburg described his seminal observation that cancer cells negotiated a switch towards anaerobic respiration from oxidative phosphorylation in return for sustained growth and evasion of normal cellular processes such as cell cycle control and apoptosis. The underlying mechanism that governs this phenomenon has yet to be firmly established. Given the centrality of mitochondria to apoptosis and bioenergetics, we proposed experiments that employed ubidecarenone as an intracellular energy ambassador wherein high-throughput genomic, metabolomic, and proteomic analyses were employed to capture the energetic and molecular signature of cancer cells. Analyses were performed on oncogenic breast, prostate, liver, pancreatic, skin (melanoma, squamous cell carcinoma) and were compared to normal fibroblasts, keratinocytes, hepatocytes, kidney, adipocytes, and human aortic and endothelial cells. The results suggested a hallmark discovery that clearly delineated a differential effect of cancer vs. normal cells under hyperglycemic, hypoxic, and lactate-stressed conditions. A decreased activation of the Pentose Phosphate Shunt was significantly downregulated in cancer cells suggestive of a shift towards utilization of oxygen and glycolysis while Bcl-2 dependent cell death mechanisms were restored. Most notably, cross-talk mechanisms related to p53 and Vegf in the cancer cells and tumor markers and pathology in animal models of melanoma and pancreatic cancer demonstrated efficacy in a topical and intravenous form of Cytotech Labs API 31510, a ubidecarenone based technology that targets the Bcl-2 protein family downstream of a cellular metabolic shift. Conversely, normal cells did not exhibit the aforementioned characteristic indicative of a cancer-specific mechanism. We propose a novel underlying mechanism for the Warburg Hypothesis and demonstrate that pre-clinical work on API 31510 in cancer suggest that it may be a viable agent for control of cancer metabolism and serve as safe, effective anti-cancer agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 990. doi:10.1158/1538-7445.AM2011-990
Abstract 994: Monitoring glucose, glutamine and fatty acid metabolism to determine metabolic vulnerability of tumor cells in vitro
Authors: Pike Lisa , Smift Amy , Ferrick David , Wu Min
Published: June 2012

Tumor cells display a metabolism that is strikingly different from that of the normal tissue from which they originated. This metabolic alteration results from activation of oncogenes and/or loss of tumor suppressor genes controlling cellular metabolic pathways as well as adaptation to their microenvironments and provides energy and cellular building blocks, allowing tumor cells to proliferate and survive. The altered metabolism of glucose and glutamine are becoming one of the hallmarks of tumor cells. We present a method that enables rapid assessment of the ability of tumor cells to catabolize the three major nutrient substrates, glucose, glutamine and fatty acids in a microplate using the XF Extracellular Flux Analyzer. Cellular oxygen consumption rate is used as an indicator of substrate oxidation by the mitochondrial respiratory chain upon substrate addition and extracellular acidification rate upon glucose addition is used as an indicator of glycolysis. Furthermore, the maximal capacity of glycolysis and of mitochondrial substrate oxidation is determined by forcing one of the two energy-producing pathways to operate at maximum. In addition, pharmacological inhibition of distinct metabolic pathways allows us to discern the biochemical pathways used, i.e., whether glutamine is metabolized through aminotransferase or glutamate dehydrogenase. Using this approach, we have demonstrated that the nutrient levels in culture medium can markedly shift the ability of cancer cells to use glucose for glycolysis versus oxidation of glutamine and fatty acids and is accompanied by modulation of cell proliferation rate. For example, excessive nutrients induced activation of fatty acid oxidation and enhanced glutamine oxidation in myc-expressing SF188 glioblastoma cells. This shift in substrate use also sensitized these cells to inhibition of fatty acid oxidation. We observed a concomitant increase in the maximal capacity of the mitochondrial respiratory chain with elevated substrate oxidation. In contrast, the apparent glycolysis rate of SF188 cells was much reduced but the glycolytic capacity was retained, which implies these cells have the flexibility to channel glucose to either energy or building block production required for cell mass duplication. We suggest that this type of dynamic analysis provides a rapid means to determine the ability of various types of tumor cells to use glucose, glutamine and fatty acids as well as the maximal capacity of the machineries of glycolysis and substrate oxidation, which may be indicative of metabolic vulnerability and therefore targets of intervention of a particular tumor cell type.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2011-994
Abstract LB-102: Inhibiting the expansion of hepatic cancer stem cells by targeting β-catenin/CBP interaction with ICG-001 in liver Pten deficient mice
Authors: Stiles Bangyan L. , Galicia Vivian , He Lina , Kahn Michael
Published: June 2012

We used a liver specific Pten (phosphatase and tensin homologue deleted on chromosome ten) deletion murine model to investigate the mechanism of hepatic cancer stem cell activation in vivo. We have shown that loss of PTEN induces the transformation of liver progenitor cells to tumor initiating cells (TICs). Activation of TIC in this model involves death of hepatocytes resulting from PTEN loss induced liver steatosis. Such hepatocytes death presents a selection pressure and altered the progenitor cell niche for mutant progenitor cells to proliferate and replace liver parenchyma. We found that hepatic injury is followed by expansion of hepatic progenitors and upregulation of hepatic progenitor cell markers. In addition, we also found factors governing the progenitor niche such as Wnt ligands including Wnt5a, 7a and 10a as well as Wnt signaling receptor Fzd are induced. Furthermore, β-catenin activation is observed in vivo as well as in progenitor cells isolated from these mice. Thus, we hypothesize that activation of Wnt serves as the niche signal to allow proliferation of mutant progenitors. To test this hypothesis, we treated the isolated progenitor cells with a small molecule compound ICG-001 to interrupt b-catenin/CBP binding. β-catenin/CBP signal allows the activation of progenitor niche by inducing selfrenewal signals while inhibiting differentiation signals. We showed that ICG-001 treatment blocked the self-renewal signals and proliferation of the isolated progenitors in vitro. The in vivo experiment is ongoing and will also be presented at the meeting. Our study demonstrated that β-catenin activation, either directly through PTEN loss or indirectly through PTEN loss induced lipotoxic death of hepatocytes, serves as an important mediator for the activation of Pten mutant progenitor cells. Targeting such niche with our inhibitor may prove to be a viable approach to inhibit liver tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-102. doi:10.1158/1538-7445.AM2011-LB-102
Abstract LB-105: Identifying molecular markers associated with chemo-resistance in ovarian cancer stem cells
Authors: Dyall Sheetal , Lawrenson Kate , Dafou Dimitra , Gayther Simon A.
Published: June 2012

Stem-like cancer cells, or cancer stem cells (CSC) have been proposed to sustain the growth of tumour cells and give rise to drug resistance in cancer. Side populations (SP) of cells, characterised by their differential ability to efflux Hoechst 33342 from their cytoplasm via multi-drug transporters, are also thought to be enriched in CSCs. The aim of this study was to identify SPs of cancer cells in epithelial ovarian cancer (EOC), and genes within SP populations that both characterize SP cell in EOC cell lines and play a role in the development of resistance to chemotherapeutic agents.

Using flow cytometry, we isolated SP cells from a panel of EOC cell lines. Detectable levels of viable SP cells were observed in the cell lines IGROV1 (1.8–5%), OVCAR3 (0.8%), OVCAR8 (0.2%), and the matched pairs of cisplatin-sensitive cell lines and their resistant variants OV2008 (2.5%)/C13*(1.5%) and A2780 (0.2%)/A2780CP (0.2%). In vitro characterization of SP cells from IGROV1, OVCAR3 and OVCAR8, compared to the non-SP cells showed that SP cells have higher colony forming efficiency when cultured as three-dimensional spheroids in non-adherent culture conditions. A proportion of single SP cells also had the ability to form multi-cellular spheroids in culture (a stem-cell ‘like’ feature) while non-SP cells did not. Under adherent culture conditions, SP cells show a range of morphologies, consisting of holoclones (which are enriched in SP cells) and paraclones (depleted of SP cells). Sub-cloning of holoclones generated both holoclones and paraclones; culturing paraclones led to the formation of only a few paraclones, which senesced. Differential whole-genome gene expression microarray profiling of SP vs non-SP cells identified the P-glycoprotein ABCB1 as the most highly upregulated gene in SP cells, consistent with other studies. Profiling of A2780 and its chemo-resistant derivative A2780CP identified Lin28, an embryonic stem cell marker, as the most differentially over-expressed gene in A2780CP SP cells. SiRNAs to ABCB1 were used to test the hypothesis that knockdown of this marker reduce proportionally the stem cell population in the IGROV1 EOC cell line; siRNAs to Lin28 were used to test whether knockdown of this gene re-sensitized A2780CP cells to cisplatin. Ongoing studies are evaluating the effects on chemosensitivity in vivo, by knocking down Lin28 in A2780CP cells expressing a luciferase reporter. Using Xenogen live animal imaging technologies, tumor burden is being evaluated in real-time, comparing Lin28 knockdown xenografted cell lines with non-silencing siRNA controls.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-105. doi:10.1158/1538-7445.AM2011-LB-105
Abstract LB-108: Formation and maintenance of mesenchymal and stem-cell states in the breast by paracrine and autocrine signals
Authors: Scheel Christina , NgEaton Elinor , Li Sophia Hsin-Jung , Chaffer Christine L. , Reinhardt Ferenc , Kah Kong-Jie , Bell George , Guo Wenjun , Rubin Jeffrey , Richardson Andrea , Weinberg Robert A.
Published: June 2012

Passage through an epithelial-mesenchymal transition (EMT) is associated with the acquisition of migratory and self-renewal abilities in human mammary epithelial cells (MECs). The signaling mechanisms that induce and then maintain these properties have remained unclear. We describe three signaling pathways, involving Transforming Growth Factor (TGF)-beta as well as canonical and non-canonical Wnt signaling, that collaborate to induce epithelial MECs to enter into a mesenchymal and SC-like state. Acting as autocrine signaling loops, these pathways then maintain migratory and self-renewal abilities in normal MECs and control tumorigenicity and metastasis in their transformed derivatives. Autocrine signaling is enabled, at least in part, through downregulation of secreted Wnt antagonists and Bone Morphogenetic Proteins. Like immortalized and transformed MECs, the maintenance of migratory and self-renewal abilities in primary human MECs is dependent on these autocrine loops, suggesting that similar mechanisms regulate biological properties of normal tissue stem cells and tumor-initiating cells in the breast

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-108. doi:10.1158/1538-7445.AM2011-LB-108
Abstract LB-109: BCR-ABL1 kinase activity but not its expression is dispensable for Ph+ quiescent stem cell survival which depends on the PP2A-controlled Jak2 activation and is sensitive to FTY720 treatment
Authors: Neviani Paolo , Harb Jason , Oaks Joshua , Walker Christopher , Santhanam Ramasamy , Paisie Carolyn , Eiring Anna , Zhang Bin , Perazzona Bastianella , Ma Y , Mao Charlene , Marcucci Guido , Holyoake Tessa , Volinia Stefano , Cortes Jorge , Caligiuri Michael , Huettner Claudia , Bittman Robert , Chen Ching-Shih , Arlinghaus Ralph , Hokland Peter , Roy Denis-Claude , Bhatia Ravi , Perrotti Danilo
Published: June 2012

Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs.

Methods: HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assays; Western blot, confocal microscopy and FACS analyses.

Results: We observed increased BCR-ABL1 expression with impaired kinase activity in quiescent CML HSCs, in which BCR-ABL1 per se is required for induction of JAK2 that subsequently activated β-catenin and inhibited PP2A. In fact, PP2A was suppressed in CML but not normal CD34+/CD38−/CD90+ HSCs. FTY720 and/or its non-immunosuppressive (S)-FTY720-OMe derivative markedly reduced survival and self-renewal of CML but not normal quiescent HSCs through BCR-ABL1 kinase-independent and PP2A-mediated JAK2 and β-catenin inhibition. Importantly, FTY720 also strongly diminished BCR-ABL1+ LT-HSC frequency in serial BM transplantation assays.

Conclusions: The pharmacologic targeting of the newly-identified BCR-ABL1 kinase-independent JAK2/β-catenin interplay in quiescent HSCs with FTY720 and its derivatives, might lead to cessation of lifelong patient dependence on TKIs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2011-LB-109
Abstract LB-11: PTEN modulates EGFRvIII-induced SHP2 phosphorylation and translocation to affect glioblastoma sensitivity to tyrosine kinase inhibitors (TKIs)
Authors: Kapoor Gurpreet Singh , Zhan Yi , Fetting Heather , O'Rourke Donald M.
Published: June 2012

Co-expression of EGFRvIII and PTEN in a small subset of recurrent glioblastoma tumors has been shown to increase sensitivity to tyrosine kinase inhibitors (TKIs). However, the exact mechanism of EGFRvIII and PTEN interaction in response to TKIs is still unresolved. Our recent work has shown that SHP2 PTPase is required for EGFRvIII-mediated transformation. The present work was aimed to study the effect of PTEN on EGFRvIII-induced SHP2 phosphorylation and translocation and modulate glioblastoma sensitivity to tarceva treatment. We show that tarceva treatment (10μM/ml) abolished EGFRvIII, EGFR, Gab1, SHP2 and Erk1/2 phosphorylation in LN229.EGFRvIII cells at all time intervals. On the contrary, phosphorylation of EGFRvIII and Erk1/2 in U87MG.EGFRvIII cells was inhibited at early time points, but was restored in 2 to 6 hours. Interestingly, phosphorylation of Akt, Gab1 and SHP2 (Tyr580) was unaffected in U87MG.EGFRvIII cells, but EGFR and SHP2 (Tyr542) phosphorylation were inhibited in a time-dependent manner. MTT proliferation and soft agar transformation assays demonstrated that U87MG.EGFRvIII cells were resistant to tarceva treatment when compared to LN229.EGFRvIII cells. Immunofluorescent labeling of U87MG.EGFRvIII cells with an anti-phospho-SHP2 (Tyr542) antibody showed membrane, cytoplasmic, perinuclear and nuclear localization of SHP2, whereas LN229.EGFRvIII cells exhibited membrane staining of phosphorylated SHP2. However, both the cell lines exhibited strong nuclear staining of phospho-SHP2 (Tyr580). Interestingly, tarceva treatment inhibited membrane localization of phospho-SHP2 (Tyr542) and nuclear localization of phospho-SHP2 (Tyr580) in LN229.EGFRvIII cells after 1 hr, but did not affect SHP2 phosphorylation and localization in U87MG.EGFRvIII cells at all time intervals. Furthermore, stable expression of PTEN in U87MG.EGFRvIII cells conferred relocalization of phospho-SHP2 (Tyr542) to the membrane. Notably, U87MG.EGFRvIII/PTEN clones showed a partially untransformed phenotype. Furthermore, phosphorylation of SHP2 (Tyr580) and Erk1/2 was totally abolished in U87MG.EGFRvIII/PTEN subclones. Our data indicate that the expression of PTEN in U87MG.EGFRvIII cells conferred a phenotype similar to LN229.EGFRvIII cells. Collectively, these observations infer that SHP2 is a downstream effecter of PTEN and that PTEN deficiency may lead to SHP2 activation and nuclear localization by EGFRvIII, which may result in increased resistance to TKIs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-11. doi:10.1158/1538-7445.AM2011-LB-11
Abstract LB-133: Golgin-160 binds to the tumor suppressor Par-4 and attenuates its secretion
Authors: Bhattarai Tripti Shrestha , Singh Jaya , Qiu Guofang , Machamer Carolyn E. , Rangnekar Vivek M.
Published: June 2012

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein that selectively induces apoptosis in cancer cells but not in normal cells. Previously thought to function exclusively by intracellular mechanisms, Par-4 is now recognized to also utilize an extrinsic pathway that is coupled to intracellular Par-4 to mediate apoptosis. Par-4 is secreted by almost all cell types, and is detected in the conditioned medium of cultured cells or in the serum of animals. Systemic Par-4 inhibits tumor growth progression, yet the precise mechanism(s) involved in the regulation of Par-4 secretion has not been characterized. Par-4 secretion is induced by an increase in endoplasmic reticulum (ER) stress; this observation is paradoxical as protein secretion is generally inhibited by elevated ER stress. Toward the goal of investigating proteins that may directly bind and regulate Par-4 secretion, we performed a yeast two-hybrid analysis, as well as co-immunoprecipitation studies. These studies consistently identified the golgi peripheral membrane protein, Golgin-160, as a binding partner of Par-4. We further noted that Golgin-160 down-modulates the secretion of Par-4 in response to ER stress caused by tunicamycin. Our data suggest that Par-4 secretion is attenuated by binding to Golgin-160. This regulatory process may have important implications in microenvironments (for example, those involving inflammation) that have increased Golgin-160, as depleted Par-4 secretion may show greater propensity for transformation and malignant progression. Since Par-4 selectively induces apoptosis in transformed cells but not in normal/non-transformed cells, strategies to down-modulate Golgin-160 to promote Par-4 secretion may have broad implications in cancer therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-133. doi:10.1158/1538-7445.AM2011-LB-133
Abstract LB-14: Low connexin-26 predicts the development of metastasis after radical prostatectomy
Authors: Bijnsdorp Irene V. , Rozendaal Laurence , van Moorselaar R. Jeroen A. , Geldof Albert A.
Published: June 2012

Prostate cancer is the second most common cause of death in males. Not all tumors will become metastatic within a patients life time, but when metastasized no curative therapy is available. It is therefore important to identify molecules that predict whether the tumor will become metastatic. Connexin-26 (Cx26) is a gap-junction protein that is involved in cell-cell contacts and in cell adhesion. Cx26 is involved as a tumor suppressor in various tumor types. The aim of the present study was to determine Cx26 expression in localized prostate cancer and relate the expression to the development of metastasis. Cx26 was stained by immunohistochemical staining in radical prostatectomy tissues of 43 patients. The expression levels were scored by a pathologist. Cx26 expression was observed in 95% of the patients. No relation was found to Cx26 expression in the tumor and the development of metastasis (p=0.2). Interestingly, a clear relation was found between low Cx26 expression in benign epithelial cells within the same sections and the development of metastasis (p<0.03). These metastases developed in about 5–40 months after radical prostatectomy. Kaplan Meier analysis showed a relation between a low Cx26 (score <75) expression in benign epithelial cells and time to PSA progression (p=0.01). Time to progression was associated with PSA and Cx26 expression in benign epithelial cells (Multivariate Cox proportional hazards analysis, p=0.05). In conclusion, Cx26 expression in the benign epithelial cells was associated with the development of metastasis. Cx26 expression could therefore be used as a predictor of outcome after radical prostatectomy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-14. doi:10.1158/1538-7445.AM2011-LB-14
Abstract LB-141: Expression and localization of Id3 in human prostate cancer
Authors: Sharma Pankaj , Patel Divya , Chinaranagari Swathi , Chaudhary Jaideep
Published: June 2012

Purpose: The inhibitor of differentiation (Id) proteins are dominant negative basic helix-loop-helix family of transcription factors that lack the DNA-binding basic domain but have intact HLH domain. The four members of the Id protein family (Id1-Id4) are known to play a critical role in the coordinated regulation of gene expression during cell growth, differentiation and tumorigenesis. Although in vitro studies have demonstrated the role of Id1 in regulating prostate cancer (PCa) cell proliferation, apoptosis and androgen independence, its clinical significance in PCa remains controversial. On the other hand there is lack of evidence on the expression of Id2 and Id3 in PCa progression. Studies have demonstrated that silencing of Id3 is significantly more effective in blocking proliferation of PCa cells as compared to Id1 whereas Id2 silencing has been primarily shown to be involved in apoptotic pathway. Hence in this study we re-evaluated the role of Id1 in PCa and extended our study to show that Id3 protein expression has better clinical relevance compared to Id1 and Id2 in PCa.

Experimental design: The expression of Id-1, -2 and -3 was determined by real time and Western blotting along with their sub-cellular localization by ICC in PCa cell lines LNCaP, DU145 and PC3. To assess their significance in prostate cancer progression, the protein expression was investigated in clinical prostate tumor samples on high density tissue microarray by IHC.

Results: The expression of Id1 and Id3 mRNA was low in LNCaP and DU145 as compared to PC3 whereas the level of Id2 expression was similar between all the PCa cell lines. The protein expression data was consistent with the mRNA levels in the PCa cell lines. All three Id's were primarily cytoplasmic with some nuclear presence. The IHC data showed a significant increase in the expression of Id1 and Id3 in clinical prostate tumor tissue samples compared to normal prostate tissue whereas Id2 showed elevated levels but was not significant as compared to Id1 and Id3. The expression of Id3 protein correlated with the Gleason grading and stage of PCa.

Conclusions: This is the first study demonstrating a correlation between increased expression of Id3 and prostate cancer progression. It also reconfirms and establishes the association of Id1with PCa. The results warrant further investigation into isoform specific effects of Id1and Id3 on prostate tumorigenesis. (Research Support: NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. The first two authors contributed equally to this work.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2011-LB-141
Abstract LB-144: I-kappa-B kinase (IKK)ε expression in pancreatic ductal adenocarcinoma
Authors: Guo Jianping , Cheng Anna , Henderson-Jackson Evita B. , Kim Donghwa , Malafa Mokenge , Coppola Domenico
Published: June 2012

I-kappa-B-kinase (IKKε) is a serine/threonine protein kinase that belongs to the IKK kinase family. Recent studies have shown that IKKε functions as a breast and ovarian cancer oncogene. Here we demonstrate frequent overexpression of IKKε in pancreatic ductal adenocarcinoma (PDA). We immunohistochemically evaluated 78 PDA using the avidin-biotin-peroxidase method and the anti-IKKε, rabbit polyclonal antibody. Elevated IKKε reactivity (IHC score 4–9) was observed in 64.1 % (50/78) PDAs, but in 0% (0/113) of non neoplastic pancreatic ductal epithelium (NMP) (p < 0.001). Kaplan-Meier analysis of overall survival (OS) revealed that patients with high IKKε-IHC scores (4–9) had a significantly shorter survival than that of patients with low IKKε -IHC scores (0–3) (Log-rang test p = 0.023), independent of either tumor stage or grade. These data indicate that deregulation of IKKε is a common event in PDA and might play an important role in the pathogenesis of this deadly disease. In addition, IKKε could serve as a prognostic marker and potential therapeutic target for PDA intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-144. doi:10.1158/1538-7445.AM2011-LB-144
Abstract LB-151: Tumor VEGF-C promotes immune suppression and tolerance
Authors: Lund Amanda W. , Raghavan Vidya , Hirosue Sachiko , Duraes Fernanda , Hugues Stephanie , Swartz Melody
Published: June 2012

The expression of the vascular endothelial growth factor, VEGF-C, is correlated with tumor invasion and hypothesized to induce metastasis through enhanced lymphangiogenesis and the creation of an “escape route”. However, new evidence suggests that VEGF-C can induce invasion in the absence of obvious tumor lymphangiogenesis, and furthermore, that lymphatic transport may play a role in modulating immunity. Still however, the role of the lymphatics as players in anti-tumor immunity and tumor invasion remains unexplored. Here we present a new model for VEGF-C induced tumor-invasion in which VEGF-C and lymphatic endothelial cells play a direct role in the suppression of anti-tumor immunity. Using a B16 F10 mouse melanoma that overexpresses VEGF-C, we demonstrated that VEGF-C induces the expression of the lymphoid homing cytokine, CCL21, within the stroma of the tumor and drives lymphocyte trafficking, fluid drainage and metastasis to the sentinel draining lymph node. Within the tumor stroma VEGF-C recruited a more regulatory immune cell infiltrate including increased regulatory T cells (CD25+FoxP3+) and M2 macrophages (CD11b+CD80-CD206+) and decreased antigen specific cytotoxic T cells (CD8+Trp2+). To test the ability of this mediated VEGF-C response to overcome host immunity we engineered a B16 F10 line to express ovalbumin (OVA) as a model tumor antigen. When we preimmunized the mice against OVA we observed that VEGF-C provided protection against vaccine (OVA/LPS) induced anti-tumor immunity. Growth of OVA bearing tumors was inhibited with vaccination and correlated with an increase in CD8+ antigen specific cytotoxic T cells. However, neither the growth nor the infiltration of cytotoxic T cells was affected by vaccination when the tumors also overexpressing VEGF-C. Additionally, to determine the fate of antigen specific CD8+ we adoptively transferred OT-I T cells by tail vein injection and observed their homing to the draining lymph nodes and tumor. In these microenvironments, when exposed to VEGF-C, adoptively transferred cells lost function over time as shown by the detection of Annexin V and decreased IFNγ expression. These results suggest that the expression of VEGF-C by a primary tumor not only promotes metastasis through the expansion of the lymphatic network but also through an immunological switch within the tumor and draining lymph node. We therefore present one of the first studies to identify an active, rather than passive, role for the lymphangiogenic growth factor, VEGF-C, and local lymphatic endothelial cells in the regulation of tumor immunity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2011-LB-151
Abstract LB-155: A new syngeneic MYCN-overexpressing neuroblastoma mouse model and MYCN-DNA vaccine
Authors: Stermann Alexander , Huebener Nicole , Lode Holger N.
Published: June 2012

High-level expression of MycN caused by amplification of the gene characterizes the malignant phenotype of neuroblastoma. Recent studies suggest that MYCN is a suitable target for immunotherapy, but up to now, a syngeneic NB mouse model over expressing MYCN is not available to examine immunotherapeutic strategies in vivo.

Here, we report the development of a tetracycline inducible MYCN expressing murine NB cell line syngeneic to A/J mice and a MYCN-DNA vaccine. For this purpose, the murine NB cell line C1300, syngeneic to A/J mice, was stably transfected with a tetracycline inducible MYCN expression vector. Stable transfection was verified by real-time PCR and Western-Blot, revealing high expression levels of MYCN RNA and protein, respectively. Furthermore, a MYCN-DNA vaccine, based on epitopes encoding for three peptides from the murine MYCN protein sequence with high affinity to the A/J mouse MHC class I allele H2-Kk, was designed and tested in vivo for its ability to induce an antigen-specific immune response. Lymphocytes isolated from A/J mice vaccinated with the MYCN vaccine effectively lysed C1300-MYCN +tet cells in cytotoxicity assays in contrast to wild type C1300 cells and lymphocytes from control mice. Lymphocytes from minigene vaccinated mice produced significantly higher amounts of IFN-γ after stimulation with irradiated C1300 cells than lymphocytes from control mice. Interestingly, vaccine induced cytotoxic T lymphocytes also kill parental C1300 tumor cells which show low MYCN expression. We expect that this effect will be enhanced when MYCN-over expressing C1300 cells are employed in a similar cytotoxicity assay which will be reported at the meeting.

In summary, we report the development of a new MYCN-DNA vaccine and a murine tetracycline inducible MYCN NB cell line providing an useful syngeneic mouse model for in vivo evaluation of MYCN directed immunotherapeutic strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-155. doi:10.1158/1538-7445.AM2011-LB-155
Abstract LB-214: Rac1 and Cdc42 GTPases as novel targets in ovarian cancer
Authors: Wandinger-Ness Angela , Kenney S. Ray , Agola Jacob , Roxby Joshua , Surviladze Zurab , Silberberg Melina , Zeineldin Reema , Vestling Anna , Bologa Cristian , Ursu Oleg , Oprea Tudor , Muller Carolyn , Lomo Lesley , Sklar Larry , Hudson Laurie G.
Published: June 2012

Epithelial ovarian cancer is the major cause of gynecologic malignancy deaths. Because of their roles in cell adhesion and migration, Rho family GTPases have been suggested as potential therapeutic targets in human cancers. We identify the Rac1 and Cdc42 GTPases as relevant targets in papillary serous and endometriod tumors. Cdc42 is overexpressed in primary human ovarian tumors and cancer cell lines, and a novel splice variant Rac1b is upregulated in tumors of advanced stage and grade. GTPase activities in primary ascites are 3 to 6-fold higher than in cultured cells. R-Naproxen was identified by high throughput screening of a Prestwick compound library as a select non-steroidal anti-inflammatory drug (NSAID) from 23 tested that selectively targets Rac1 and Cdc42 in a bead-based assay using purified proteins. The drug is demonstrated to have positive benefit against cell behaviors required for ovarian cancer dissemination and metastasis using both cell lines and primary human tumor cell isolates. Human ovarian cells show slowed cell proliferation, as well as impaired migration, adhesion and invadopodia formation. Other NSAIDs with structural similarity (S-naproxen and 6-methoxy naphthalene acetic acid) lack these properties, while a specific Rac inhibitor NSC 23766 mimics the effects. Molecular docking shows R-Naproxen can bind the GDP-bound, but not GTP-bound Rac1, suggesting it may act by stabilizing Rac and Cdc42 in the inactive state. R-Naproxen has potential for rapid translation and efficacy in the treatment of metastatic ovarian cancer on account of FDA approval and novel activities against Rho-family GTPases.

Funding for this study was generously provided by NIH grants U54MH074425, U54MH084690, R03MH081231, P30CA118100 and UNM Cancer Center FIG.0990MD.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2011-LB-214
Abstract LB-243: Functional oncogene identification in hematologic malignancies
Authors: Weigert Oliver , Lane Andrew , Kopp Nadja , Rodig Scott , Yoda Akinori , Weinstock David M.
Published: June 2012

Next generation sequencing of hematologic malignancies has identified tens to hundreds of mutations in coding sequence per sample. However, the functional significance of nearly all of these mutations remains unclear. Importantly, systematic analyses to determine the phenotypic effects of somatic mutations from cancer samples have proven cumbersome and low-yield. We developed an alternate approach to efficiently identify functionally-important, cryptic alterations directly from hematologic malignancy specimens. In this system, retroviral cDNA libraries are constructed from patient samples and then used to infect the IL3-dependent B-cell line, BaF3. Expression of oncogenic kinases, transcription factors and other oncogenes results in IL3-independent survival, allowing for the isolation of individual transformed clones and sequencing of integrated cDNA. Streamlined methods for cDNA cloning and retroviral transduction have made it both feasible and cost-efficient to construct patient-specific libraries for oncogene identification. With this approach, cryptic mutations, translocations and other rearrangements with functional significance can be identified. We previously performed functional oncogene screening in a panel of precursor B-cell acute lymphoblastic leukemia (B-ALL) specimens with normal karyotypes. From our initial screen, we recovered an IL3-independent BaF3 clone that harbored a full-length mutant version of the cytokine receptor subunit CRLF2. We subsequently demonstrated that 5–10% of adult and pediatric B-ALL harbor CRLF2 alterations that frequently coincide with activating mutations in Janus kinase 2. With support from Stand Up To Cancer, we applied functional screening to 6 additional hematologic malignancy subtypes. We supplemented screening with limited exome sequencing of angioimmunoblastic T-cell lymphomas, biphenotypic acute leukemia and BALL to confirm the presence of functional alleles and recover possible cooperating mutations. With this approach, we identified a panel of alterations in these diseases and confirmed several to be somatic and functionally relevant, including novel mutations in JAK2, TYK2, IDH2 and FLT3. In addition, targeted sequencing identified the same nonsense mutation in an alpha kinase in multiple cases of angioimmunoblastic T-cell lymphoma. In conclusion, large-scale sequencing efforts are underway at many centers to identify somatic mutations in hematologic malignancy samples. Screening of retroviral cDNA libraries offers an efficient alternative, focused on phenotypically meaningful, and therefore potentially drugabble, alterations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-243. doi:10.1158/1538-7445.AM2011-LB-243
Abstract LB-263: Novel tumor suppressors and mutationally affected signaling pathways in small-cell lung cancer identified by whole exome sequencing
Authors: Peifer Martin , Thomas Roman K.
Published: June 2012

Small-cell lung cancer (SCLC) is an aggressive lung cancer subtype characterized by early metastasis and a peculiar neuroendocrine phenotype. Unfortunately, SCLC is difficult to sample because these patients are not routinely undergoing surgery. In order to shed light on the biology of this tumor type and to discover novel therapeutic targets, we have conducted whole-exome sequencing of 24 SCLC tumors as well as high-density gene copy number analyses of 64 SCLC specimens. On average we observe a high mutation rate of 7.9±1.0 protein changing mutations per million basepairs and an unusually high C:G>A:T transversion rate that has been associated with excessive smoking. In addition to frequent mutations in TP53 (80%) and RB1(63%), we identified mutations in LRP1B (13/24 samples), a gene that is known to be frequently mutated in lung adenocarcinoma. To our surprise, we found a significant enrichment of neuronal genes (FDR q-value: < 10e-10) suggesting that the neuroendocrine differentiation of SCLC might be primarily driven by mutational events. Other frequently mutated genes encode proteins involved in cell-cell and cell-matrix interactions. This observation might explain the aggressiveness and metastatic capability of SCLC. A systematic analysis of copy number losses and loss of heterozygosity revealed a distinct pattern for novel candidate tumor suppressor genes when integrated with somatic mutations. Performing the pathway analysis on each individual sample clearly showed that SCLC tumors segregate by mutational landscape. Furthermore, the analysis of autopsy-derived tumor specimens revealed that surgically resected tumors are not dissimilar to late-stage SCLC. In summary, we have defined the mutational and genomic landscape of SCLC that may help explaining some of the phenotypic and clinical properties of this fatal tumor type. Our data provides a rich source for future functional analyses of putative tumor suppressor genes and oncogenes as well as for potential novel drug targets.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2011-LB-263