bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1143: EGF induces epithelial to mesenchymal transition and cancer stem-like cell properties in human head and neck cancer cells via promoting Warburg Effect
Authors: Xu Qilin , Zhang Qunzhou , Le Ahn D.
Published: August 2015

Head and neck cancer is one of the 10 most common cancers worldwide. Over-expression of EGFR has been demonstrated in approximately 90% of head and neck cancers, which is associated with progressive tumor growth, metastasis, recurrence, and resistance to chemo- and radiation therapies. Recurrent tumors are often therapy-resistant and may have their origin in resistant cancer stem cells (CSCs) and/or in tumor cells with an epithelial-mesenchymal transition (EMT) phenotype. Recently it is increasingly accepted that metabolic changes in cancer cells can drive tumor formation and progression.

In our study, we used 20ng/ml EGF to treat human tongue cancer cell line, SCC-1 cells. Our results demonstrated that EGF induces EMT in oral SCC cells as characterized by a downregulation of E-cadherin (an epithelial marker), an upregulation of vimentin (a mesenchymal marker), increased invasive and metastatic capacities, and acquisition of stem-like properties such as the increased expression of ALDH1 and enrichment of CD44+/CD24− proportion of cancer cells. We also found that EGF treatment concomitantly enhanced L-lactate production and blocking glycolysis by 2-DG robustly reversed EGF-induced EMT and CSC-like phenotypes of OSCC cells. Then we further demonstrated that EGF regulates glycolysis/EMT/CSCs axis through downstream EGFR/PI3K signaling pathways in an EGFR/PI3K-dependent but AKT-independent manner. Importantly, we showed that in vivo treatment with 2-DG remarkably reduced the expression of ALDH1 and vimentin at both the in situ tumors and regional lymph nodes.

Taken together, these findings have demonstrated that EGF drives tumor growth and metastasis through induction of EMT and CSC generation, which is orchestrated by an enhanced glycolytic metabolic program in OSCC cells. This work was supported by National Institute of Health Research Grant, R01DE 019932, Oral and Maxillofacial Surgery Foundation (OMSF) Research Grant, and the Schoenleber funding support

Citation Format: Qilin Xu, Qunzhou Zhang, Ahn D. Le. EGF induces epithelial to mesenchymal transition and cancer stem-like cell properties in human head and neck cancer cells via promoting Warburg Effect. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1143. doi:10.1158/1538-7445.AM2015-1143
Abstract 1147: Differential expression of enzymes associated with glycine metabolism in ovarian cancer stem like cell
Authors: Shin So-Jin , Chung Hye-Won , Lee Hyun-Gyo , Choi Eun Som , Cho Chi-Heum
Published: August 2015

OBJECTIVES: The recent advances in understanding the relation between cancer and metabolism have highlighted the relevance of serine/glycine biosynthesis and one-carbon metabolism. The aim of this study was to evaluate the expression of enzymes associated with serine/glycine metabolism in ovarian cancer stem like cells (CSCs) and discuss their potential clinical implications.

METHODS: We established primary ovarian cancer cell cultures from dissected fresh tumor tissue. To isolate CD117(+) and CD44(+) CSCs from primary ovarian cancer cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity. In the present study, we used Western blotting and immunohistochemistry to examine three serine-/glycine-metabolism-associated proteins (PHGDH, SHMT, and GLDC) in CSCs and ovarian cancer cases using tissue microarray (TMA). The expression of stemness gene (Nanog, Oct3/4, Sox2 and ABCG2) mRNA was determined by RT-PCR and protein expression was detected by Western blot analysis.

RESULTS: GLDC, PHGDH and SHMT, associated with serine/ glycine metabolism, showed lower expression in the CSCs. TMA showed that the different expressions of GLDC, PHGDH and SHMT in platinum-sensitive and platinum-resistant ovarian cancer cases (p<0.004). GLDC mRNA was most frequently expressed in primary ovarian cancer culture cells. By Cox multivariate analysis, tumor SHMT1 negativity (HR: 2.132, 95% CI: 1.229-3.664, p = 0.007) was associated with short overall survival.

CONCLUSIONS: Based on our findings, we concluded that the expression of serine-metabolism-associated proteins was related with resistance of ovarian cancer cells and patient's survival.

Citation Format: So-Jin Shin, Hye-Won Chung, Hyun-Gyo Lee, Eun Som Choi, Chi-Heum Cho. Differential expression of enzymes associated with glycine metabolism in ovarian cancer stem like cell. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1147. doi:10.1158/1538-7445.AM2015-1147
Abstract 1148: Ang-(1-7) decreases HIF-1α and migration of oral squamous cell carcinoma
Authors: Guimaraes Andre Luiz Sena , Fraga Carlos Alberto de Carvalho , Farias Lucyana Conceição , Guimarães Talita Antunes , De Paula Alfredo Maurício Batista , Mota Amanda , Santiago Lais , Gomez Ricardo Santiago , Santos Sergio Henrique Sousa , Santos Robson Augusto Souza dos , Bader Michael
Published: August 2015

induced by cobaltous chloride and the underlying mechanism.

Methods and Results

We first confirmed that Ang-(1-7) decreases HIF-1α signaling in a newly developed hydroxypropyl-β-cyclodextrin (HPBCD)-based Ang-(1-7) nano-formulation in a novel transgenic rat model of inducible insulin resistance and DM2. The chronic administration of this compound prevented the marked elevation of HIF-1α in diabetic rat kidney. Next, mouse embryonic fibroblasts (MEFs) and SCC-9 cells were incubated in a well humidified incubator with 5% CO2 and 95% air at 37 °C (normoxic condictions). For hypoxic cultures, cells were incubated 150 μM CoCl2 for 24 hours and maintained in a 5% CO2 atmosphere at 37°C. Then, we divided cells into 4 groups including control, CoCl2, Ang-(1-7) and CoCl2 + Ang-(1-7). HIF-1α protein expression and AKT phosphorylation were decreased in hypoxic cells after treatment with Ang-(1-7). Besides, Ang-(1-7) decreased SCC-9 cell migration in hypoxic condition.

Conclusions

In conclusion, our current findings suggest that Ang-(1-7) attenuates HIF-1 α protein stabilization via AKT and Mas receptor-dependent mechanism. In addition, we found that the Ang-(1-7)/Mas receptor inhibit oral cancer cell migration.

Note: This abstract was not presented at the meeting.

Citation Format: Andre Luiz Sena Guimaraes, Carlos Alberto de Carvalho Fraga, Lucyana Conceição Farias, Talita Antunes Guimarães, Alfredo Maurício Batista De Paula, Amanda Mota, Lais Santiago, Ricardo Santiago Gomez, Sergio Henrique Sousa Santos, Robson Augusto Souza dos Santos, Michael Bader. Ang-(1-7) decreases HIF-1α and migration of oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1148. doi:10.1158/1538-7445.AM2015-1148
Abstract 1150: mTORC2 enhances flux through the hexosamine biosynthetic pathway by regulation of GFAT1 expression
Authors: Jacinto Estela , Moloughney Joseph , Lynch Thomas , Wu Chang-Chih , Ibironke Olufunmilola , Navia Aixa , Chou Po-Chien , Zhang Sisi , Rabinowitz Joshua , Werlen Guy
Published: August 2015

Metabolic and biosynthetic pathways drive cell growth and proliferation in response to nutrients and growth factors. Highly proliferating cells utilize glucose and glutamine to fuel biosynthetic processes. These two nutrients serve as substrates for the glutamine:fructose-6-phosphate amidotransferase (GFAT1), the rate-limiting enzyme in the hexosamine biosynthetic pathway (HBP), which ultimately produces UDP-GlcNAc that is necessary for protein glycosylation. Despite a role for the HBP in insulin resistance and lifespan extension, the mechanisms underlying GFAT1 regulation in vivo has remained elusive. We found mTOR complex 2 (mTORC2) controls flux through the HBP via regulation of GFAT1 expression levels in response to glucose. In the absence of mTORC2, GFAT1 expression is reduced and highly sensitive to glucose starvation. Furthermore, UDP-GlcNAc is highly diminished and glycosylation of specific transmembrane proteins such as CD147 is defective upon mTORC2 disruption. However, mTORC2 is also required for glycolysis and other biosynthetic pathways whose metabolites feed into the HBP. Thus, although exogenous UDP-GlcNAc can partially rescue glycosylation defects, it does not rescue the metabolic deficiencies in the absence of mTORC2. Like GFAT1, key enzymes of biosynthetic pathways have decreased expression in mTORC2-disrupted cells. Thus, by regulating levels of metabolic enzymes, mTORC2 coordinates flux through biosynthetic pathways in response to glucose availability. Our findings have implications for therapeutic targeting of mTORC2 in insulin resistance and cancer metabolism.

Citation Format: Estela Jacinto, Joseph Moloughney, Thomas Lynch, Chang-Chih Wu, Olufunmilola Ibironke, Aixa Navia, Po-Chien Chou, Sisi Zhang, Joshua Rabinowitz, Guy Werlen. mTORC2 enhances flux through the hexosamine biosynthetic pathway by regulation of GFAT1 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1150. doi:10.1158/1538-7445.AM2015-1150
Abstract 1155: Regulation of glutamine metabolism: Allosteric activation and inhibition of mitochondrial glutaminase
Authors: Stalnecker Clint , Ulrich Scott , Erickson Jon , Ramachandran Sekar , DeBerardinis Ralph , Cerione Rick
Published: August 2015

The hydrolysis of glutamine to glutamate plus ammonia, catalyzed in the mitochondria by the enzyme glutaminase (Gls), plays a crucial role in the metabolism of glutamine by cancer cells. Recently, we discovered a novel class of allosteric inhibitors of a splice variant of Gls, known as glutaminase C (GAC), which inhibits the growth of Rho-GTPase transformed cells without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we took advantage of mouse embryonic fibroblasts (MEFs) transformed by the induced expression of an oncogenic guanine nucleotide exchange factor (GEF) named Dbl, which hyper-activates the Rho GTPases Rac, Rho, and CDC42, together with 13C-labeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis. We then determined how 968 inhibits the catalytic activity of GAC using biophysical approaches on purified GAC enzyme. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrated how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays, together with novel GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity for monomeric GAC, and that the dose-dependent binding directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. We then investigated the 968 structure-activity relationship, and to this end, characterized a robust real-time direct binding and inhibition assay for testing a panel of 968 derivatives. Additionally, we have developed novel 968-derivatives with unique functional moieties, such as fluorescent and photo-cross linking groups, to further elucidate the mechanism of GAC inhibition using recombinant GAC in vitro as well as in cancer cells. Together, these findings represent recent advances in our understanding of the regulation of GAC activity both in vitro and in cells, and highlight the potential future of targeting glutamine metabolism with novel small molecule therapeutics.

Citation Format: Clint Stalnecker, Scott Ulrich, Jon Erickson, Sekar Ramachandran, Ralph DeBerardinis, Rick Cerione. Regulation of glutamine metabolism: Allosteric activation and inhibition of mitochondrial glutaminase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1155. doi:10.1158/1538-7445.AM2015-1155
Abstract 1159: Ketosis and hyperbaric oxygen elicit potent anti-cancer effects in vitro and in vivo
Authors: Poff Angela , Ward Nathan , Seyfried Thomas , D'Agostino Dominic
Published: August 2015

The Warburg effect and tumor hypoxia underlie a unique cancer metabolism characterized by glucose dependency and abnormal mitochondrial function. We have previously shown that this phenotype can be exploited therapeutically by glucose restriction with the ketogenic diet, dietary ketone supplementation, or hyperbaric oxygen (HBOT). We hypothesized that combining these therapies would be an effective non-toxic treatment regimen, so we tested this combination therapy in the VM-M3 mouse model of metastatic cancer. Treated mice exhibited decreased tumor growth rate, decreased metastatic spread to the lungs, kidneys, spleen, adipose, and liver, decreased liver tumor vascularization, and lived twice as long as controls. To further characterize the effects of these therapies, we measured proliferation, viability, ROS production, and expression of key signaling molecules in VM-M3 cells exposed to the following conditions: high glucose (control; 25mM), low glucose (LG; 3mM), ketone supplementation (βHB; 5mM), hyperbaric oxygen (HBOT; 90 min, 2.5 ATA), or combination therapy (LG+BHB+HBOT). Proliferation of VM-M3 cells was measured with trypan blue hemocytometry and was significantly decreased from controls at 24, 48, 72, and 96 hours. Viability of VM-M3 cells was measured by fluorescence microscopy with calcein and EthD-1 and was decreased by 19.2% in LG, 12.5% in βHB, and 38.2% in LG+βHB+HBOT treated cells compared to control. ROS (superoxide) production was measured by fluorescent microscopy with DHE and was increased by 46% in HBOT and by 48% in LG+βHB+HBOT treated cells. Treatment with βHB significantly decreased expression of HIF-1α and pAkt compared to control as measured with western blot. HBOT also inhibited HIF-1α expression but increased pAkt expression in vitro. This study strongly supports further investigation into mechanisms of action and the use of these metabolic therapies as a potential cancer treatment.

Citation Format: Angela Poff, Nathan Ward, Thomas Seyfried, Dominic D'Agostino. Ketosis and hyperbaric oxygen elicit potent anti-cancer effects in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1159. doi:10.1158/1538-7445.AM2015-1159
Abstract 1163: Phosphoserine aminotransferase 1 (PSAT1) as a novel anti-tumor target in hepatocellular carcinoma
Authors: Ding Zhaobing , Wee Kenneth , Kim Eung-Sam , Samanta Animesh , Steckel Michael , Raschke Marian , Velan Sendhil , Chang Young-Tae , Haegebarth Andrea , Ziegelbauer Karl , Gruenewald Sylvia , Han Weiping
Published: August 2015

With an estimated 700,000 new cases per year, hepatocellular carcinoma (HCC) represents the fifth commonest cancer and is the third leading cause of cancer death worldwide. Its 5-year survival rate after surgery is only 5-6%. Although HCC cases occur predominantly in the East and Southeast Asia and Sub-Saharan Africa, the incidence is rising in the West, largely due to an increasing incidence of Hepatitis C virus infection, alcoholic liver disease, and non-alcoholic steatohepatitis related to obesity and type II diabetes. Besides sorafenib, an oral multikinase inhibitor approved for the treatment of HCC, there is no other pharmacological agent in the treatment of HCC.

Cancer cells undergo dramatic metabolic reprogramming to sustain uncontrolled proliferation. Accumulating evidence supports abnormal metabolic pathways as an emerging hallmark of cancer. To identify metabolic genes required for HCC tumorigenesis, we compared gene expression profiles of a rat orthotopic and a mouse DEN (Diethylnitrosamine)-induced HCC model to a liver regeneration model and to normal liver by PCR arrays, transcriptomic analysis, and metabolomic profiling. Serine and glycine levels were increased in tumors of the HCC models as determined by NMR spectroscopy. PSAT1 (phosphoserine aminotransferase 1), an enzyme in the serine biosynthesis pathway, was identified as one of the top up-regulated genes in the HCC models. An increase in PSAT1 protein levels in tumor samples versus normal liver tissue was confirmed by Western blot. Knockdown of PSAT1 in rat hepatoma cells (MH3924a) led to a dramatic reduction in cell proliferation in vitro and in vivo. Moreover, a doxycycline-inducible knockdown approach confirmed the anti-proliferation effect of PSAT1 depletion. Interestingly, depletion of PSAT1 showed a variable effect on other HCC cells (e.g. Hep3B and Huh-7), suggesting the involvement of other genetic and metabolic factors. Detailed analysis of PSAT1 as a potential drug target for HCC is currently being addressed in in vivo studies.

Citation Format: Zhaobing Ding, Kenneth Wee, Eung-Sam Kim, Animesh Samanta, Michael Steckel, Marian Raschke, Sendhil Velan, Young-Tae Chang, Andrea Haegebarth, Karl Ziegelbauer, Sylvia Gruenewald, Weiping Han. Phosphoserine aminotransferase 1 (PSAT1) as a novel anti-tumor target in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1163. doi:10.1158/1538-7445.AM2015-1163
Abstract 1169: Growth regulation of bladder cancer cells by compounds that affect glucose metabolism
Authors: Lea Michael A. , desBordes Charles
Published: August 2015

The observation that cancer cells tend to exhibit enhanced rates of glycolysis presents a target for therapeutic regulation of cancer cell growth by molecules that regulate glucose metabolism. We have extended our earlier studies on colon cancer cells with an examination in bladder cancer cells of the action of an established inhibitor of PFKFB3, namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) as a single agent and in combination with other molecules that affect glycolysis and proliferation. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB-3) is an enzyme regulating glycolysis in many tumor cells. Cell growth was monitored by staining with sulforhodamine B. Of the 8 cell lines examined, growth was most rapid in the T24 and UM-UC-3 bladder cancer cells. The slowest growth was seen with RT4, TCCSUP and HT1197 cells while intermediate growth rates were observed with 5637, HT1376, and SW780 cells. Glucose uptake from the medium and acidification of the medium over a 72-hour incubation tended to correlate with growth rate. Thus the greatest changes were seen with the T24 and UM-UC-3 cell lines and the least change was seen with RT4 and TCCSUP cells. Growth was inhibited when glutamine was omitted from the medium but growth was blocked in the absence of glucose except for RT4 cells in which growth was inhibited more in the absence of glutamine. Treatment with 10 μM 3PO as a single agent for 72 hours caused average growth inhibition in the range of 25-50% without apparent relationship to the control growth rates of the cell lines. Additive inhibitory effects on growth were observed when cells were coincubated with 3PO and either 1 mM 2-deoxyglucose, 50 μM phenformin or 1 mM butyrate. In addition to being growth inhibitory, butyrate also caused significant induction of alkaline phosphatase activity in some cell lines. When cells were incubated with phenformin as a single agent, there was generally an increased acidification of the medium that was diminished by coincubation with 3PO. In conclusion, 3PO was an inhibitor of proliferation of human bladder cancer cells and may be a useful agent in combination with other drugs that inhibit bladder cancer cell proliferation.

Citation Format: Michael A. Lea, Charles desBordes. Growth regulation of bladder cancer cells by compounds that affect glucose metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1169. doi:10.1158/1538-7445.AM2015-1169
Abstract 1174: Exploiting glioma stem cell metabolism to abrogate radioresistance in glioblastomas
Authors: Spehalski Elizabeth I. , Saito Keita , Kramp Tamalee , Tandle Anita , Rath Barbara , Cherukuri Murali , Camphausen Kevin
Published: August 2015

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and, even with aggressive treatment that includes surgical resection, radiation, and temozolomide administration, prognosis is poor due to tumor recurrence. There is evidence that within GBMs a small number of glioma stem cells (GSCs) exist, which are thought to be radiation resistant and may be capable of repopulating a tumor after treatment. Like most cancers, GBMs largely employ aerobic glycolysis to create ATP, a phenomenon known as the Warburg effect. Experiments have shown that the cellular metabolism of GSCs differs from that of differentiated cells, making them an attractive target for novel therapeutic approaches. Much work has been done to analyze the metabolic profiles of GSCs with the goal of identifying potential therapeutic targets, but little data exists linking metabolic changes to radiation resistance. The purpose of this study is to determine the impact of radiation on the cellular metabolism of GSCs and to identify changes that can be exploited for treatment in conjunction with radiation therapy.

To this end, we analyzed three human GSC lines harboring unique sets of genetic mutations that were hypothesized to differentially impact cellular metabolism. Experimental approaches include in vitro examination of metabolic rates and mitochondrial stress of the GSC lines via Seahorse extracellular flux analysis with and without radiation administration as well as analysis of tumor xenografts in mice via 13C MR spectroscopic imaging using hyperpolarized [1-13C] pyruvate as a tracer. Additionally, we are employing genomic and proteomic profiling of the GSC lines both in culture and in vivo to categorize metabolic changes following radiation treatment.

We show that the basal metabolic rates of GSC lines with distinctive genetic mutations can be differentiated using Seahorse analysis. This is confirmed in vivo by 13C MR spectroscopic imaging, thus revealing a potential non-invasive diagnostic modality. The underlying mechanism behind these metabolic differences and how this relates to each GSC's response to radiation is currently being examined. We expect that this data will result in the identification of molecular targets within the metabolic pathways of cells that can be exploited with drugs in conjunction with radiation therapy to improve the prognosis and survival of patients presenting with GBMs in the clinic.

Citation Format: Elizabeth I. Spehalski, Keita Saito, Tamalee Kramp, Anita Tandle, Barbara Rath, Murali Cherukuri, Kevin Camphausen. Exploiting glioma stem cell metabolism to abrogate radioresistance in glioblastomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1174. doi:10.1158/1538-7445.AM2015-1174
Abstract 1196: Enhancement of anti-melanoma effect of BRAF and MEK inhibition by metformin
Authors: Zhang Yanping , Peng Guangyong , Hsueh Eddy C.
Published: August 2015

Introduction: Metformin has been shown to inhibit human cancer cell growth via activation of MAP kinase with resultant inhibition of mTOR signaling pathway. Recent clinical evidence demonstrated the anti-melanoma effect of combined mutant BRAF inhibition and MEK inhibition. We determined the effect of metformin in combination with mutant BRAF and MEK inhibition on melanoma proliferation and the possible mechanisms.

Methods: Human melanoma cells (SK-23, Mel888, Mel1861) were treated with Metformin (1-20 mM), PLX4032 (a mutant BRAF inhibitor, 1-20 uM), AZD6244 (a MEK inhibitor, 1-20 uM), or combination. Cell proliferation assay was performed using Cell Titer Blue assay. Western blotting was performed to determine the expression of PARP, caspases-9, LC3A/B,Beclin1, pERK, pMEK, AMPKa, p70S6, and p4E-BP1. GAPDH was used as internal control. Autophagy was further analyzed with immunofluorescence with LC-3 and NECN1 anti-antibody for visualization of lysosomal morphology and evaluated under the Nikon Eclipse Ti microscope (20×). DAPI was used to counter stain the nucleus. Apoptosis was further analyzed with flow cytometry using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p< 0.05 was considered to be statically significant.

Results: There was dose-dependent inhibition of melanoma cell proliferation with either metformin(M), PLX4032(P), and AZD6244(A). Addition of M to PA combination had synergistic effect on the anti-melanoma proliferative effect of PA. Significantly increased expression (p<0.05) of apoptosis markers (PARP and caspases-9) and autophagy markers (LC3B and Beclin1) were observed in cells treated with PAM compared with PA. Enhanced induction of autophagy with the triple drugs was confirmed by visualization of autophagosome and autolysosome formation on immunofluorescence. Synergistic effect on apoptosis was confirmed on Annexin V assay. The PAM combination treatment also lead to significant enhancement of AMPK activation and increased phosphorylation of AMPKα at Thr-172 compared with metformin alone (p<0.01). The addition of M to PA combination resulted in inhibition of mTOR signaling with decreased phosphorylation of p70S6K and 4E-BP1 in treated cancer cells, when compared with PA or M alone.

Conclusion: We have observed a synergistic effect on suppressing human melanoma proliferation with the addition of metformin to combined inhibition of mutant BRAF and MEK. The synergism is due to enhanced inhibition of mTOR signaling pathways.

Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Enhancement of anti-melanoma effect of BRAF and MEK inhibition by metformin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2015-1196
Abstract 1197: Selenium-binding protein 1-mediated tumor suppression is associated with alterations of lipid/glucose metabolic pathways in vivo
Authors: Ying Qi , Ansong Emmanuel , Diamond Alan M. , Lu Zhaoxin , Bie Xiaomei , Yang Wancai
Published: August 2015

Selenium-binding protein 1 (SBP1) is significantly decreased in most of human cancers including liver, prostate, breast, lung and colorectal cancers, and the decreased SBP1 is associated with poor clinical outcomes and recurrence, showing a tumor suppressor role. However, the underlying mechanisms are still unclear. In this study, we constructed a doxycycline inducible SBP1 plasmid (pRetroX-Tight- Pur-SBP1) and established a stable colorectal cancer cell line HCT116-TetSBP1, then injected the HCT116-TetSBP1 cell line into BALB/c CD-1 Nu female mice subcutaneously on the right flank, the control cell line HCT116-Act was injected in the left flank. The mice was supplied with Doxycycline (200 μg/mL) in drinking water. Four weeks after injection, the tumors from both sides were isolated and the weight (gm) and volume (mm3) of tumors were determined. Tumors were stored at -80°C until used for immunoblot and isobaric tags for relative and absolute quantitation (iTRAQ) analysis. We found that induction of SBP1 showed significant inhibition of cancer cell growth and metastasis in mice. iTRAQ analysis showed 132 differentially expressed proteins in response to SBP1 induction, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism. Although all seven proteins related to glucose metabolism (GAA, GAPDH, ALDH2, Thioredoxin, ENO3, UGDH and Lumican) were downregulated by SBP1 induction, thirteen lipid metabolism-related proteins act differentially on SBP1-medited signal transduction (five proteins were upregulated: DKK1, HSP60, DHCR7, ANXA4 and AGPAT5, and eight proteins were downregulated: LPCAT2, GPX5, GAPDH, NME2, ALDH2, BPIFA3, PPIA and FABP4). Bioinformatic analysis showed that these proteins were involved in Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. Taken together, our results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.

Note: This abstract was not presented at the meeting.

Citation Format: Qi Ying, Emmanuel Ansong, Alan M. Diamond, Zhaoxin Lu, Xiaomei Bie, Wancai Yang. Selenium-binding protein 1-mediated tumor suppression is associated with alterations of lipid/glucose metabolic pathways in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2015-1197
Abstract 1205: Identification of a novel adjuvant therapeutic agent for obesity related pancreatic cancer
Authors: Harbuzariu Adriana , Garrison Robin C. , Daley-Brown Danielle S. , Beech Derrick J. , Cason Frederick D. , Harmon Tia L. , Yang Lily , Gonzalez-Perez Ruben R.
Published: August 2015

PURPOSE: Pancreatic cancer (PC) is an aggressive disease that develops in a symptom-free manner and is usually advanced at the time of diagnosis; the overall 5 year survival rate is less than 5%. Obesity is pandemic in the US and strongly linked to higher incidence of many cancer types. Obese people are 20% more likely to develop PC. We hypothesize that high level of leptin shown by obese PC patients could increase tumor progression and drug resistance. METHODS: A panel of PC cell lines (Panc-1, BxPC-3 and MiaPaCa-2) was challenged with leptin, a specific leptin inhibitor coupled with nanoparticles (IONP-LPrA2) and various chemotherapeutic agents (Gemcitabine, Cisplatin, Paclitaxel and Sunitinib). Cell viability, proliferation and the expression levels of molecular markers were analyzed. Further, we investigated the ability of PC cells to form tumorspheres when treated with leptin and IONP-LPrA2. The number of colonies was counted and pancreatic cancer stem cells (PCSC) markers (ALDH1, CD44, CD24, ESA) and OB-R expression was assessed. PCSC markers were determined in PC tissue arrays. RESULTS: PCSC markers were expressed in obese PC patients. Leptin induced S phase progression, overexpression of Notch and PCSC markers and increased stemness and tumorigenesis of PC cells. In addition, leptin impaired the efficacy of chemotherapeutic agents on PC cells and derived tumorspheres. The inhibition of leptin signaling via IONP-LPrA2 abrogated PC growth, reduced PCSC markers expression and enhanced chemotherapeutic action by inducing apoptosis. Our data suggests that leptin signaling is essential for PC cells proliferation and survival and reduces chemotherapeutic effects. These findings are of great importance for obese PC patients which show the highest level of leptin, poor prognosis and chemotherapy outcome. LPrA2 has no toxicity and it has been shown that it does not affect health status, appetite, body weight or energy balance in mice. IONP-LPrA2 could be a novel adjuvant for PC treatment, especially for obese PC patients. Acknowledgements: this work was supported by NIH/NCI 5SC1CA138658-05 and the DOD Breast Cancer Idea Award (W81XWH13-1-0382) to RRGP; NIH/2G12RR003034-26 and U54 MSM/UAB Cancer Center Partnership. This research was also supported by facilities and support services at Morehouse School of Medicine (NIH 1C06 RR18386).

Citation Format: Adriana Harbuzariu, Robin C. Garrison, Danielle S. Daley-Brown, Derrick J. Beech, Frederick D. Cason, Tia L. Harmon, Lily Yang, Ruben R. Gonzalez-Perez. Identification of a novel adjuvant therapeutic agent for obesity related pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1205. doi:10.1158/1538-7445.AM2015-1205
Abstract 1207: Nrf2 suppression results in growth inhibition and enhances response to chemotherapy and radiation therapy in triple-negative breast cancer cells
Authors: Yang Yuzhe , Yee Douglas
Published: August 2015

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional activator that specifically binds to the Antioxidant Response Element (ARE) to initiate expression of various anti-oxidative and anti-inflammation genes to protect cells from oxidative stress. Recently, Nrf2 has been shown to redirect glucose and glutamine into anabolic pathways to promote cell proliferation. Constitutive stabilization of Nrf2 has been observed in many human cancers and confers chemo- and radio-resistance of cancer cells. We examined Nrf2 expression and function in triple negative breast cancer cells (TNBC). mRNA and protein expression of Nrf2 were higher in the TNBC cell lines MDA-MB-231 and MDA-MB-436 compared to immortalized breast epithelial cell lines (MCF10A and MCF12A) and estrogen receptor positive breast cancer cell lines (MCF-7 and T47D). shRNA knock-down of Nrf2 in MDA-MB-231 and MDA-MB-436 TNBC cells showed decreased mRNA expression of multiple Nrf2 regulated anti-oxidant (SLC7A11, NQO-1, GCLC, GCLM, SLC7A5, etc) and pentose phosphate pathway genes (G6PD, PGD, ME-1, TKT, TALDO1, PPAT, etc), enhanced basal levels of cellular reactive oxygen species, impaired mitochondrial function as determined by measuring mitochondrial potentials in living cells, and reduced S phase entry. Cells with decreased Nrf2 had reduced cell growth in monolayer and anchorage independent growth assays. In addition, Nrf2 suppression reduced cell migration. Nrf2 down-regulated MDA-MB-231 cells also showed increased response towards ionizing radiation and mitomycin C treatment in clonogenic and soft agar assays. Furthermore, reduced Nrf2 expression decreased the number of stem-like (CD44+/CD24-) cells in MDA-MB-231 cell populations. Thus, Nrf2 regulated many aspects of the malignant phenotype in TNBC and suggest that inhibition of Nrf2 might be a therapeutic target for TNBC.

Citation Format: Yuzhe Yang, Douglas Yee. Nrf2 suppression results in growth inhibition and enhances response to chemotherapy and radiation therapy in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2015-1207
Abstract 1210: Biopterin metabolism drives tumor progression
Authors: Rabender Christopher , Alam Asim , Waters Mike , Mikkelsen Ross
Published: August 2015

Previous studies demonstrate that nitric oxide synthases (NOS’s) can become uncoupled during inflammatory disease states. A hallmark of this uncoupling is superoxide production from NOS in lieu of nitric oxide (NO) resulting from a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2 ratio). As BH4 is a necessary cofactor for NO production from NOS, when this ratio is reduced, NOS becomes a peroxynitrite synthase enhancing the pro-inflammatory/pro-survival tumor environment. Our lab demonstrated that NOS is uncoupled in a variety of tumor cells both in vitro and in vivo, as well as in paired human colorectal samples due to a reduced BH4:BH2 ratio and that recoupling NOS in via exogenous BH4 both in vitro and in vivo leads to decreased tumor cell proliferation. To examine the mechanism behind the reduced BH4:BH2 ratio we used the DSS/AOM model of colitis and colon tumorigenesis. Here we demonstrate that BH4:BH2 ratio is reduced in mouse colon tumors vs mouse normal colon tissue, 1.36±0.35 vs 7.1±0.6. We determined that this reduced ratio is due at least in part to direct oxidation of BH4 to BH2, as treatment of colon cells with an NADPH Oxidase inhibitor increased the BH4:BH2 two-fold; however, this is unable to account for the degree of uncoupling associated with tumor cells. Given that inflammation appears to drive NOS uncoupling, we extracted colons from DSS/AOM treated animals and preferentially harvested colonic epithelial cells and colonic tumor cells at different stages of inflammation to assess the molecular and biochemical changes in molecules important in the regulation and production of BH4. The molecules we focused on were: GTP cyclohydrolase-1 (GCH1, rate limiting enzyme in de novo BH4 synthesis), GTP cyclohydrolase 1 feedback regulatory protein (GCHFRP, regulates GCH1 expression), quinoiddihydropteridine reductase (QDPR, necessary for salvage pathway) and sepiapterin reductase (SR, necessary for both de novo synthesis and salvage of BH4). Transcript levels of these molecules in DSS treated epithelial cells showed that GCH1 and SR were upregulated but QDPR was significantly downregulated when compared with untreated cells, potentially leading to an increase in total BH4 but once oxidized to BH2, a decrease in the salvage back to BH4. Our current research investigates the protein levels as well biochemical activity of these molecules in DSS treated epithelial cells, as well as tumor cells compared with normal cells to further understand the molecular component of the decrease in BH4:BH2 ratio in tumors.

Note: This abstract was not presented at the meeting.

Citation Format: Christopher Rabender, Asim Alam, Mike Waters, Ross Mikkelsen. Biopterin metabolism drives tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1210. doi:10.1158/1538-7445.AM2015-1210
Abstract 1211: Metabolite assays to illuminate cellular energy networks
Authors: Sobol Mary , Vidugiriene Jolanta , Leippe Donna , Vidugiris Gediminas , Zhou Wenhui , Cali James
Published: August 2015

Every organism requires cellular energy metabolism to meet developmental needs. When cells change from a resting to a proliferative state, the metabolic pathways are reprogrammed to meet the demands of proliferation. Diseases such as cancer can occur when changes in the metabolic pathways support unwanted functions such as uncontrolled proliferation.

Assays for analyzing metabolites and metabolic enzymes are needed to understand cellular metabolic requirements and energy network regulation under normal and disease conditions.

We exploited the robustness, sensitivity and broad dynamic range of bioluminescence detection to develop assays for the key metabolites lactate, glucose, glutamate and glucose-6-phosphate. The technology involves using specific dehydrogenases to couple metabolite levels to NAD(P)H production. In the presence of NAD(P)H, a novel proluciferin is converted to a substrate for luciferase resulting in light signals proportional to the starting metabolite concentration. Samples (e.g. mammalian cells, culture media, tissues, 3D microtissues) are added to the metabolite detection reagent in a 1:1 ratio. An inactivation solution added to complex biological samples lowers the background and increases sensitivity by lysing the cells, inactivating cellular enzymes, and destroying endogenous NAD(P)H.

The sensitivity (1-5 pmol/sample) and broad assay window (maximum signal-to-background > 100 fold) of the assays allows simultaneous detection of multiple metabolites from the same set of samples. The wide assay window allows detection of very small changes in metabolite concentration, an advantage over conventional fluorescent and colorimetric detection methods. The assays adapt to various formats (96, 384-well plates) and require no instrumentation other than a luminometer.

The assays can be used to measure the levels of critical metabolites and to monitor the effects of inhibitors and activators of glycolysis. The shift from oxidative phosphorylation to glycolysis can be monitored by measuring lactate production and secretion as well as glucose consumption from the same set of samples. The assays easily multiplex with cell viability assays enabling analysis of drug effects on cell proliferation, cell metabolism and mitochondria function.

Citation Format: Mary Sobol, Jolanta Vidugiriene, Donna Leippe, Gediminas Vidugiris, Wenhui Zhou, James Cali. Metabolite assays to illuminate cellular energy networks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1211. doi:10.1158/1538-7445.AM2015-1211
Abstract 1220: MDM2 promotes p53-independent breast cancer metastatic phenotypes
Authors: Gao Chong , Kundu Nandini , Bargonetti Jill
Published: August 2015

Many human cancers overexpress the E3 ubiquitin ligase MDM2. The cancer genome atlas (TCGA) found that increased MDM2 expression is one of four gene pathways that correlate with all breast cancer subtypes. An increasing body of literature strongly suggests that overexpression of MDM2 can drive breast oncogenesis in the presence of mutant p53 (mtp53), as well as through molecular pathways that do not require wild-type p53 degradation. Therefore, the canonical wild-type p53 degradation pathway by MDM2 is clearly not the only important pathway. MDM2 expression has been shown to influence breast cancer cell invasion and metastasis. One potential mechanism for this is through the activation of SLUG (a transcription repressor of E-Cadherin) and the process of E-cadherin degradation. To investigate the p53-independent influence of MDM2 we engineered human breast cancer clones with inducible shRNA to knockdown MDM2 in MDA-MB-231 (mtp53 R280K), T47D (mtp53 L194F) and MCF-7 (wild-type p53) cells. Wound healing assays showed that MDM2 knockdown in all three cell lines reduced cell migratory capacity without altering p53 protein levels. In T47D.shmdm2 cells, a decrease in SLUG expression was observed with MDM2 knockdown indicating SLUG is a p53-independent target of MDM2. The knockdown of MDM2 also decreased slug and mmp9 mRNA detected by quantitative RT-PCR. To determine the biological significance of MDM2 in a system to recapitulate breast cancer architecture we examined T47D.shmdm2 cells in a 3-dimensional (3D) cell culture model. Importantly, the depletion of MDM2 in T47D.shmdm2 cells grown in 3D culture promoted luminal clearance. Our findings suggest that MDM2 can influence SLUG and MMP9 levels associated with breast cancer metastasis through a mechanism that does not require degradation of wild-type p53. Moreover our work suggests that MDM2 may promote changes to breast cancer architecture that drive metastasis.

Acknowledgements. This work was supported by a grant to JB from the Breast Cancer Research Foundation.

Citation Format: Chong Gao, Nandini Kundu, Jill Bargonetti. MDM2 promotes p53-independent breast cancer metastatic phenotypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1220. doi:10.1158/1538-7445.AM2015-1220
Abstract 1222: p53 downregulates CRKL proto-oncogene protein through miR-200 family
Authors: Sasaki Yasushi , Tamura Miyuki , Kobashi Kenta , Takeda Kousuke , Nakagaki Takafumi , Idogawa Masashi , Tokino Takashi
Published: August 2015

The downregulation of the miR-200 family has been involved in the progression and metastasis of human cancers. The miR-200 family includes two clusters: miR-200b, miR-200a and miR-429 (miR-200b-200a-429) located on chromosome 1; and miR-200c and miR-141 (miR-200c-141) located on chromosome 12. We identified response elements located around both clusters that are responsive to p53 family. Based on the similarity of their seed sequences, miR-200b, miR-200c and miR-429 have identical seed sequences, whereas the seed sequence of miR-141 and miR-200a is similar, but not identical, suggesting that miR-200b, miR-200c and miR-429 share similar mRNAs target. Here, in silico miRNA target prediction identified the v-crk sarcoma virus CT10 oncogene homolog-like (CRKL) as a potential target for miR-200b, miR-200c and miR-429 (miR-200b/200c/429). miR-200b/200c/429 inhibits the expression of CRKL mRNA and protein by directly targeting its 3′-UTR region. Importantly, endogenous CRKL expression was decreased in cancer cells with introduction of wild-type p53 and TAp63γ. Oncomine database shows that CRKL levels are significantly overexpressed in multiple cancer types compared with the corresponding normal tissues. We also found that CRKL was overexpressed in primary breast cancer tissues harboring mutant p53 genes. Our results indicate that miR-200b/200c/429, a p53-inducible miRNA, is a negative regulator of the CRKL oncoprotein and suggest a new p53-miR-200-CRKL pathway in carcinogenesis.

Citation Format: Yasushi Sasaki, Miyuki Tamura, Kenta Kobashi, Kousuke Takeda, Takafumi Nakagaki, Masashi Idogawa, Takashi Tokino. p53 downregulates CRKL proto-oncogene protein through miR-200 family. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1222. doi:10.1158/1538-7445.AM2015-1222