bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1119: Interplay between EGFRvIII and c-Met in glioblastoma multiforme
Authors: Saunders Vanessa , Ling Yuan Yuan , Anderson Jessica , Duckett Derek
Published: June 2012

Glioblastoma multiforme (GBM) is a highly invasive and lethal brain tumor which is associated with a high recurrence rate after surgery and an increased resistance to irradiation and chemotherapies. Consequently, patients suffering from GBM have a poor prognosis and a high mortality rate with an average life expectancy of less than 15 months. Key signaling components, especially receptor tyrosine kinases (RTKs), are aberrantly activated in GBM. The most frequent is the epidermal growth factor receptor (EGFR) and here the most commonly activated form, EGFRvIII, augments GBM invasiveness, promotes therapy resistance and connotes poor prognosis. There are five EGFR approved inhibitors and interestingly clinical studies have indicated that targeting the activity of the EGFR in association with conventional GBM therapies does little to inhibit tumor growth. The underlying mechanistic reasoning for this poor response are not understood and may reflect EGFR-induced trans-activation of other RTKs such as the hepatocyte growth factor receptor (c-Met), or uncoupling of EGFR signaling from its downstream mediators. Several studies have indicated that EGFR can trans-activate c-Met and consequently the dual targeting of both EGFR and c-Met significantly decreases the growth of EGFRvIII expressing tumor xenografts, indicating that inhibiting both EGFR and c-Met activity may prove beneficial in GBM therapy. Currently, the only known ligand for c-Met is the hepatocyte growth factor (HGF). Thus we hypothesize that EGFR induced HGF production is critical for the increased tumorgenicity observed in EGFRvIII expressing GBM cells. Using GBM cell lines we show that c-Met activation is increased in GBM cells that both stably and transiently express EGFRvIII. Furthermore, conditioned media taken from GBM cells expressing EGFRvIII can activate c-Met signaling in GBM cells that lack EGFRvIII. In both instances we see that the kinase activity of the EGFRvIII partially contributes to the production of HGF and the activation of c-Met in GBM cells. Aberrant c-Jun terminal kinase signaling (JNK) can be detected in the majority of patient GBMs. Using GBM cells stably lacking JNK2α2 we identify JNK2α2 signaling as a potential mechanism behind EGFRvIII activation of c-Met via the production of HGF in EGFRvIII GBM expressing cells. Taken together our data describes a novel role for JNK2α2 in mediating GBM tumorgenicity through the regulation of HGF production and the subsequent activation of c-Met signaling in GBM.

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 1119. doi:10.1158/1538-7445.AM2011-1119
Abstract 1122: Insulin receptor expression and survival of patients with non-small cell lung cancer
Authors: Kim Jin-Soo , Kim Edward S. , Liu Diane , Lee J. Jack , Solis Luisa , Behrens Carmen , Lippman Scott , Hong Waun Ki , Wistuba Ignacio I. , Lee Ho-Young
Published: June 2012

Purpose: The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with non-small cell lung cancer (NSCLC).

Methods: A total of 459 patients who underwent curative resection of NSCLC were studied (median follow-up duration, 4.01 years). Expression of the IR and IGF-1R protein in tumor specimens was assessed immunohistochemically using tissue microarrays.

Results: The cytoplasmic IR score was higher in patients with adenocarcinoma (ADC) than in those with squamous cell carcinoma (SCC) whereas cytoplasmic IGF-1R was higher in patients with SCC than those with ADC. Neither IR nor IGF-1R expression was associated with sex, smoking history, or clinical stage. Patients with positive IR or IGF-1R expression levels had poor recurrence-free (3.8 vs. 3.3 years; 3.8 vs. 2.0 years, respectively) but similar overall survival durations. Patients with high expression levels both IR and IGF-1R had shorter recurrence-free and overall survival compared to those with low levels of IR and/or IGF-1R expression. IGF-1R and IR expressions were negatively correlated with survival duration of patients with ADC and SCC, respectively. Finally, a multivariate analysis revealed the impact of IR, but not IGF-1R, as an independent prognostic predictor of survival: hazard ratio (HR) for OS, 1.005 (95% confidence interval [CI], 1.001 – 1.010], HR for RFS, 0.608 (95% CI, 1.001 – 1.009) when tested as a continuous variable.

Conclusions: Overexpression of IR, but not IGF-1R, appears to foretell a poor survival among patients with NSCLC, especially those with SCC. Thus, expression of IR has a negative prognostic value and future clinical trials with therapy interventions of IR regulation should be under the consideration for this patient population.

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 1122. doi:10.1158/1538-7445.AM2011-1122
Abstract 1125: The insulin-like growth factor (IGF) axis is involved in normal prostate differentiation and downregulated in local prostate cancer
Authors: Massoner Petra , Rennau Michael Ladurner , Heidegger Isabel M. , Kloss-Brandstätter Anita , Schäfer Georg , Seifarth Christof , Klocker Helmut
Published: June 2012

Introduction and Objectives: Clinical trials targeting the insulin-like growth factor 1 receptor (IGF1R) in different tumors, including breast cancer, colorectal cancer, leukemia (ALL, CML), non-small-cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer and sarcomas are underway. Encouraging data from phase II studies in NSCLC and sarcomas led to rapid development of phase-III studies. However, a phase-III study with an anti-IGF1R antibody combined with erlotinib in advanced NSCLC was terminated recently for safety reasons and lack of efficiency (ClinicalTrials.gov Identifier: NCT00673049). Have we possibly moved to early to clinical trials without entirely understanding the role of the IGF axis in normal and cancer development?

The IGF1R is part of a tightly regulated molecular network named IGF axis. The IGF axis is implicated in tumor development and progression, but also exerts essential functions in normal tissues, including control of growth processes, tissue homeostasis, differentiation and metabolism.

Methods: Here we analyzed the mRNA expression level of the all members of the IGF axis, including IGF1, IGF2, IGFBP1-IGFBP6, IGF1R, IGF2R and INSR in microdissected tissue specimens of local prostate cancer. We compared IGF expression levels in malignant and benign prostate areas in both, the epithelial and the stromal compartment. Furthermore we investigated whether IGF expression levels are changed during normal prostate differentiation and describe a novel cellular model for prostate differentiation.

Results: We show that the IGF axis is not up- but downregulated in local prostate cancer, in both the epithelial and the stromal compartment. Using cellular models we demonstrate that the IGF axis is induced during normal prostate epithelial differentiation and confirm herewith that the IGF axis is required for tissue homeostasis and normal epithelial differentiation in the prostate.

Conclusions: Our data confirm a pivotal role of the IGF axis during normal prostate differentiation, which may overcomes its pro-oncogenic effect in local prostate cancer. Treatments of local prostate cancer with IGF1R inhibitors cannot be advised based on these data.

Source of Funding: COMET center Oncotyrol and Tyrolean Cancer Foundation.

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 1125. doi:10.1158/1538-7445.AM2011-1125
Abstract 113: Aberrant DNA methylation of the HIF-1α inhibitor gene CITED4 in breast cancer
Authors: Huang Katie T. , Takano Elena A. , Mikeska Thomas , Byrne David J. , Dobrovic Alexander , Fox Stephen B.
Published: June 2012

CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain, 4 (CITED4) inhibits the interaction between HIF-1α and p300/CBP thereby reducing expression of downstream hypoxia responsive genes. Since we have previously shown that diminished levels of nuclear CITED4 as a consequence of down-regulation or translocation to the cytoplasm are correlated with HIF-1α elevation in breast cancer, we hypothesized that either hypermethylation or somatic mutation of the CITED4 gene may also underlie elevated HIF-1α levels in breast tumors.

CITED4 DNA methylation and mutation were analyzed in 168 breast carcinoma samples and 23 cancer cell lines using methylation-sensitive high resolution melting (MS-HRM) and DNA sequencing, respectively. Although no somatic CITED4 mutation was found, there was evidence of CITED4 promoter region methylation in five breast carcinoma samples, and three breast cancer cell lines (heterogeneously methylated in Hs578T and MDA-MB-231, and fully methylated in MDA-MB-453). There was a significant association between low CITED4 mRNA and high methylation in Hs578T (P = 0.0001) and MDA-MB-231 (P < 0.0001) and no expression was found in fully methylated cell line MDA-MB-453.

Methylated CITED4 breast cancer cell lines were treated with 5-aza-2-deoxycytidine (DAC) and/or trichostatin A to evaluate the regulation of CITED4 expression through DNA methylation. CITED4 expression was re-expressed in fully methylated MDA-MB-453 cell line and increased expression in MDA-MB-231 (21.9-fold) and Hs578T (4.9-fold) after DAC mediated demethylation, and treatment with trichostatin A alone also resulted in increased CITED4 expression. A synergistic effect was seen after the combined treatment of DAC and trichostatin A.

Furthermore, re-expressed CITED4 expression after DAC treatment may reduce HIF-1α and its downstream genes activity. MDA-MB-231 breast cancer cell line treated with DAC under hypoxia (0.1% oxygen) and demonstrated the downregulation of HIF-1α downstream genes VEGFA and SLC2A1 (P = 0.0029) when compared with the untreated MDA-MB-231 cell line under hypoxia. Immunohistochemstry also demonstrated that HIF-1α and downstream CA9 levels had decreased after CITED4 re-expressed under the hypoxic condition from 23% of positive staining in control to 10% after DAC treatment for HIF-1α and from 5 % to 3% for CA9.

These data suggest that, CITED4 gene expression can be epigenetically silenced through DNA methylation in breast cancer, which may enhance the activity of HIF in developing aggressive breast cancer phenotypes.

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 113. doi:10.1158/1538-7445.AM2011-113
Abstract 1130: mPGES-1 drives the oncogenic potential of EGFR signaling
Authors: Finetti Federica , Erika Terzuoli , Donnini Sandra , Giachetti Antonio , Ziche Marina
Published: June 2012

Prostaglandins, mostly PGE-2, have assumed an important role in cancer biology. Regarding of PGE-2 production, recent investigations have focused on PGE-2 synthases (mPGEs-1, mPGEs-2 and cPGEs), specifically on mPGEs-1, the only inducible enzyme among those so far identified in cells. Whereas cPGEs and mPGEs-2 are constitutively expressed at relatively low levels, mPGEs-1 is highly inducible by pro-inflammatory stimuli. mPGEs-1 upregulation has been detected in many epithelial tumors, and its silencing has been reported to reduce preneoplastic lesions. Despite the evidence linking mPGEs-1 and tumorigenesis, most studies investigating the inducible nature of the enzyme in cultured cells have been performed using a variety of inflammatory stimuli, while little is known about the behavior of the enzyme in tumor cells challenged with oncogenic stimuli. In this work we investigated mPGES-1 regulation in cultured epithelial tumor cells exposed to epidermal growth factor (EGF), an oncogene product which, through its receptor, EGF-receptor, is responsible for tumorigenisis in a wide array of solid tumors. We used three cell lines, representative of colon (HT-29), epidermoid (A431) and lung (A549) tumors, examining the expression of mPGEs-1 protein following EGF-receptor activation by EGF. We found that cells responded to EGF-receptor stimulation with a transient surge of the transcription factor Egr-1, its translocation from cytosol to necleus, an increase of mPGEs-1 mRNA and protein, as well as, an enhanced PGE-2 formation. These changes were suppressed by silencing Egr-1 and by EGF-receptor blockade or by inhibitors of the EGF-receptor-dependent mediators, such as MAP-kinase, ERK1/2. Moreover, cell activation through EGF-receptor induced a tumorigenic phenotype, detectable by clonogenic assay, which regressed when mPGEs-1 was silenced. We also explored the effect of inhibiting the EGF-receptor in A431 xenografts on mPGEs-1 expression and tumor growth, finding a parallel decline of both.

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 1130. doi:10.1158/1538-7445.AM2011-1130
Abstract 1139: Ras association domain family protein 1, isoform C (RASSF1C) promotes breast tumor growth in vivo
Authors: Reeves Mark E. , Aragon Robert J. , Alfakouri Mariana , Chen Shin-Tai , Amaar Yousef G.
Published: June 2012

Breast cancer will kill more than 40,000 women in the United States this year, second in cancer deaths only to carcinoma of the lung. Therefore, a better understanding of the molecules and pathways that lead to breast cancer development is important because it could lead to specific therapies that interrupt these pathways. Recently, the Ras association domain family 1 (RASSF1) gene was identified as a Ras effector that plays an important role in carcinogenesis. The RASSF1 gene encodes two major isoforms, RASSF1A and RASSF1C, derived by alternative promoter selection and alternative mRNA splicing. While RASSF1A is extensively studied and is an established tumor suppressor, very little is known about RASSF1C function

Our laboratory is interested in determining the role of RASSF1C in human cancer cell growth. We have previously shown that of RASSF1C promotes cell proliferation and migration and attenuates apoptosis of breast cancer cells in vitro. To confirm our hypothesis that RASSF1C may be a growth promoter, we have tested the growth of human breast cancer cells over-expressing RASSF1C in nude mice. Our preliminary studies show that breast cancer cells over-expressing RASSF1C developed significantly larger tumors compared to control cells. We have confirmed the expression of RASSF1C in tumor tissue using both RT-PCR and immune-staining analyses. Together, our previous in vitro and current in vivo findings further support our hypothesis that RASSF1C, unlike RASSF1A, is not a tumor suppressor and rather it appears to play a role in stimulating proliferation and survival of breast cancer cells.

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 1139. doi:10.1158/1538-7445.AM2011-1139
Abstract 1159: Identification of potential miRNA biomarkers for breast cancer using TaqMan® Pri-miRNA assays
Authors: Liang Yu , Markoy Athina , Lianidou Evi
Published: June 2012

Identification of cancer biomarkers depends on measuring the differential genotypes and phenotypes between normal and disease tissues. However, cancer is characterized by accumulation of genetic lesions and heterogeneity, and hence great efforts need to be invested to distinguish driver versus passenger abnormalities. Several strategies have been employed to achieve the distinction, including conservation of genetic lesions between mouse models and human diseases, gain of copy number/amplification, deletion/loss of heterogeneity, and epigenetic alterations. Redundancy of some microRNA (miRNA) genes has been an interesting finding and its biological significance is not clear. These redundant miRNA loci have different genomic locations and are transcribed independently, and yet are processed into identical mature miRNA, and these loci are annotated as -1, -2, -3, etc, following the miRNA gene name. Selective expression of these loci could be a regulatory strategy to control expression of other genomically associated genetic elements that are critical to the disease processes, while the required level of mature miRNA can still be maintained by leveraging the redundancy. The proof-of-principle of this rationale was to compare between normal breast and breast cancer cell lines the expression of miRNA genes that have multiple loci and are associated with other genetic elements such as other miRNA genes within the same clusters or their host protein-coding/non-coding genes. This investigation narrowed down to mir-100 gene that is clustered with let-7a-2 gene, both of which lost expression completely in three luminal-type breast cancer cell lines but not in one basal-type cell line and normal breast, whereas expression from let-7a-1 and let-7a-3 remains unchanged between normal breast and cell lines tested. Loss of mir-100 and let-7a-2 expression appears to be caused at least by loss of one copy or both copies of these two loci. Deletion of mir-100 locus can also be detected in clinical specimens. These results suggest that this method might effectively distinguish driver versus passenger differentially expressed genes.

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 1159. doi:10.1158/1538-7445.AM2011-1159
Abstract 1172: The impact of siRNA targeting Colon Cancer Associated Transcript 1 (CCAT-1) on proliferation of HT-29 cells
Authors: Tsivin Victoria , Halle David , Gure Ali O. , Pavlov Vera , Roistacher Marina , Ilyiayev Nadia , Mitrani-Rosenbaum Stella , Stojadinovic Alexander , Trink Barry , Rubinstein Abraham , Nissan Aviram
Published: June 2012

Introduction: Colon cancer Associated Transcript-1 (CCAT-1) is a non-coding RNA transcript highly expressed in colorectal cancer (CRC) but not in normal colonic tissue. The role of CCAT-1 in tumorigenesis is yet to be defined. Small interfering RNAs (siRNA) are a group of sequence-specific posttranscriptional gene silencing molecules. Recent advances in the design and delivery of targeting siRNA molecules allows efficient and highly specific gene silencing in mammalian systems. The most common application of siRNA gene-silencing in oncology is to identify loss-of-function phenotypes in genes that will result in decreased proliferation or death of cancer cells.

Aim: To study the role of colon cancer associated transcript 1 (CCAT-1) in proliferation of human colon cancer cells using RNA interference.

Methods: Small interfering RNAs (siRNA) targeting CCAT-1 were transfected into HT-29 colon cancer cells. At day one, 80×105 HT-29 cells per well were plated and suspended in RPMi 1640 medium. At day two, 50 pmol CCAT-1 siRNA and 0.5 µl lipofectamine 2000 were added to each well. CCAT-1 expression was studied by real-time PCR in order to evaluate gene silencing. Cell proliferation was studied by MTT incorporation assay. Twenty four hrs after transfection, 50 µl of MTT were added to the cells media and incubated for 4 h at 37° C. The purple Formazan product, converted MTT, was dissolved by the addition of 150µl/well DMSO and OD readings were obtained at 550 nm.

Results:By examining AV average OD readings at 550 nm, untreated HT-29 cells (10 wells), cells treated with lipofectamine 2000 (10 wells) and cells silenced by siRNA of CCAT-1 (10 wells) showed a significant decrease in proliferation: 2.4±0.64, 1.54±0.63, 0.8±0.14 respectively (p=0.002). Furthermore, comparing AV OD readings between 24 and 48 h in the silenced cells, showed a decrease of 38% in proliferation. This was compared to untransfected cells and cells treated with lipofectamine 2000 which showed an increase of 39% and 22% respectively. Twenty four hrs after transfection, real time PCR results showed a reduction in CCAT-1 RNA levels of 70% compared to controls and 48 hrs after transfection, CCAT-1 RNA levels were reduced to 55% of baseline.

Conclusions: Our results may support a role for CCAT-1 in the regulation of colon cancer cell proliferation.

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 1172. doi:10.1158/1538-7445.AM2011-1172
Abstract 1175: Persistent upregulation of U6 small RNA in the serum of breast cancer patients
Authors: Appaiah Hitesh , Goswami Chirayu , Mina Lida , Badve Sunil , Sledge George , Liu Yunlong , Nakshatri Harikrishna
Published: June 2012

Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of disease. Sera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21, and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III), and the small nucleolar RNA44 (SNORD44), were also analyzed for normalization. Sera from additional 14 healthy and 15 patients with overt metastasis were examined for U6, miR-16, and miR-195 levels. SNORD44 and miR-16 did not vary significantly in the sera of patients and healthy volunteers and thus served as normalization controls. Unexpectedly, we observed upregulation of U6 (2.4 fold, p=0.001) and 5S RNAs (1.7-fold, p=0.02) but not any of the other microRNAs analyzed in the sera of patients without active disease. U6 level was significantly elevated in the sera of patients with overt metastasis compared to healthy women. Elevated serum U6 levels in patients who are clinically cancer-free suggest persistently altered systemic RNAP-III activity in cancer patients and that cancer cells may not be the only primary source of serum U6. Additionally, U6 and 5S, to some extent, are of limited value for normalization.

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 1175. doi:10.1158/1538-7445.AM2011-1175
Abstract 1195: EphA4 is a novel tumor suppressor in lung adenocarcinoma
Authors: Peng Shaohua
Published: June 2012

Lung cancer is the leading cause of death from cancer, but the mechanisms behind its initiation and progression are not completely understood. Receptor tyrosine kinases (RTKs) are important in normal cellular physiology as well as in the pathogenesis of a variety of tumors. In this study, we explored the roles of the human Eph kinase family in lung adenocarcinoma. The Eph fmaily is the largest RTK superfamily with 14 receptors and 8 ligands (ephrins). Eph receptors have been implicated both as tumor suppressors and oncogenes. To explore the effect of Eph receptors and ephrins on lung cancer, we analyzed gene expression and survival data from 432 lung adenocarcinomas from Director's Challenge Consortium by univariate Cox proportional hazards model. Of the 11 Eph receptors and 8 ephrins analyzed, only the expression of EphA4, EphA5, EphB2, ephrinA1, and ephrinB3 correlated with overall survival. Analysis of an independent dataset confirmed that EphA4 and ephrinA1 expression negatively correlated with survival in lung adenocarcinoma. After adjusting for age, gender and stage, low EphA4 expression remained significantly associated with a poor outcome for both overall and relapse-free survival with a hazard ratio of 1.57. To validate the gene expression profile data, we measured EphA4 expression in 9 NSCLC cell lines and found that the expression levels of EphA4 by microarray were well correlated with expression levels measure by qPCR and Western blotting (R2=0.89, R2=0.79 respectively), which indicate that the tissue microarray probesets are of high quality. To investigate the role of EphA4 as a putative tumor suppressor, we overexpressed EphA4 in NSCLC cell lines with low basal expression and found that the cell migration was inhibited by 38% and E-cadherin expression was increased. Likewise, E-cadherin expression was decreased with EphA4 knockdown in NSCLC cell lines with high basal EphA4 expression. These data are consistent with a role for EphA4 as a tumor suppressor via increased expression of the cell surface adhesion protein E-cadherin and concomitant decreased cell migration.

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 1195. doi:10.1158/1538-7445.AM2011-1195
Abstract 12: JAK2V617F drives Mcl-1 expression and sensitizes acute myeloid leukemia cells to dual inhibition of JAK2 and Bcl-XL
Authors: Guo Jun , Roberts Lisa , Kraft Jeffrey B. , Merta Philip J. , Glaser Keith B. , Shah Omar J.
Published: June 2012

Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 and STAT5 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I), attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in JAK2V617F-harboring acute leukemia lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-XL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-XL/-2 is synergistic in the presence of the JAK2V617F mutation. These findings suggest that JAK/Bcl-XL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating JAK2 mutations.

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 12. doi:10.1158/1538-7445.AM2011-12
Abstract 1251: Anti-cancer mechanisms of se compounds: A stable isotope-resolved metabolomic (SIRM) approach
Authors: Fan Teresa WM , Lorkiewicz Pawel , Higashi Richard M. , Lane Andrew N.
Published: June 2012

Recent rapid advances in analytical tools, especially NMR and mass spectrometry, allow an unprecedented range of metabolites and their transformation pathways to be determined, which transcends classical targeted metabolic studies. We have coupled these tools with stable isotopic tracers [1-3] to investigate the anticancer efficacy and mechanism(s) of different selenium forms using lung adenocarcinoma A549 cells as a model. Selenite (SeO3), methylseleninic acid (MSA) and selenomethionine (SeM) induced distinct changes in steady-state concentrations and 13C labeling patterns of various metabolites including amino & organic acids, nucleotides, antioxidants, as well as phospholipids (PL) and PL metabolites. These changes were determined using a combination of multi-nuclear 2-D NMR, GC-MS, and FT-ICR-MS techniques, which enabled perturbations in multiple metabolic pathways to be simultaneously reconstructed for relating to phenotypic changes. At low µM levels, both SeO3 and MSA inhibited growth, caused cytoskeleton disintegration, and reduced the mitochondrial redox potential in A549 cells. SeM was growth-stimulatory at ≤ 10 µM and inhibited growth but only at 40-fold higher concentration and the extent was much less than MSA or SeO3. These phenotypic differences were related to the early inhibition of glutaminolysis, Krebs cycle, and protein biosynthesis by SeO3, rapid inhibition of pyruvate carboxylation by MSA. SeM had few comparable metabolic effects. Subsequent inhibition of PL biosynthesis was also more substantial in MSA and SeO3 than in SeM-treated cells. The protein and gene expression for key enzymes in these pathways were probed to discern altered metabolic regulations. Thus, the anti-cancer action of MSA differed from that of SeO3 while SeM was not effective against lung cancer cells. The SIRM approach facilitated the resolution of the complex, form-specific anticancer mechanism of Se compounds and is generally applicable to other cancer research problems.

1. Fan, T., et al., Metabolomics-edited transcriptomics analysis of Se anticancer action in human lung cancer cells. Metabolomics Journal, 2005. 1(4): p. 325-339

2. Lane, A.N. and T.W.-M. Fan, Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY. Metabolomics, 2007. 3: p. 79-86.

3. Lane, A.N., et al., Isotopomer analysis of lipid biosynthesis by high resolution mass spectrometry and NMR. Analytica Chimica Acta, 2009. 651: p. 201-208.

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 1251. doi:10.1158/1538-7445.AM2011-1251
Abstract 1254: Altered lipid and glucose metabolism is a cellular adaptation to tumor acidic microenvironments
Authors: Wojtkowiak Jonathan W. , Barkey Natalie M. , Kumar Virendra , Lloyd Mark C. , Gatenby Robert A. , Gillies Robert J.
Published: June 2012

As a consequence of increased glucose metabolism and poor perfusion, solid tumors are acidic. Tumor cells must overcome this physical microenvironmental barrier in order to progress. To better understand phenotypes associated with extracellular acidosis, MDA-MB-231 human breast carcinoma cells were acutely and chronically exposed to low pH (6.7) growth conditions. In the acute phase, low pH reduced proliferation, induced a G1cell cycle arrest, and increased cytoplasmic vacuolization, with no observable effect on cell viability. Following chronic (3 month) exposure, MDA-MB-231 cells had restored proliferation to control levels with no cell cycle arrest, yet remained highly vacuolated. Our prior work identified a subset of these vacuoles to be autophagic, while the rest appeared to be lipid droplets. Confocal microscopy of MDA-MB 231 cells stained with Nile Red, an intracellular fluorescent lipophilic dye, identified the cytoplasmic vacuoles as lipid aggregates. A significant number of lipid aggregates accumulated in low pH treated MDA-MB-231 cells (p < 0.01), and this was also observed in a series of breast and non-breast cancer cell lines, suggesting that this is a universal response to extracellular acidosis. To investigate the source of the lipid, it has been observed that accumulation is serum dependent; indicating that it is either dependent on growth factors or comes from an exogenous (albumin) source. To investigate a de novo mechanism, high resolution NMR spectroscopy was used to measure 13C enriched lactate isotopomers following metabolism of D-[1,2-13C] glucose. These analyses showed that glucose flux through the pentose phosphate pathway was three-fold higher in low pH cells, compared to controls, representing a major shift in glucose metabolism from Embden Meyerhoff to the Pentose Phosphate pathway, which results in increased production of NADPH, necessary for de novo lipid synthesis. Autofluorescence microscopy showed an altered distribution of NAD(P)H under acidic conditions. Furthermore, C75, a fatty acid synthase inhibitor, induced significant cytotoxicity in low pH treated MDA-MB-231 cells, but not controls. This is further indicative of a de novo mechanism. These data suggest that the cellular survival response to extracellular acidosis depends on lipid accumulation and profound changes in glucose metabolism.

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 1254. doi:10.1158/1538-7445.AM2011-1254
Abstract 1255: Role of choline in adaptive mechanisms in choline phospholipid metabolism of human breast cancer cells
Authors: Krishnamachary Balaji , Mori Noriko , Gadiya Mayur , Mironchik Yelena , Glunde Kristine , Bhujwalla Zaver M.
Published: June 2012

Proton magnetic resonance spectroscopy studies of cancer have consistently detected increased cellular phosphocholine (PC) and total choline-containing compounds (tCho), which are closely related to malignant transformation, invasion and metastasis [1]. Enzymes in choline metabolism present attractive targets that can be exploited for treatment [2]. Two of these enzymes, choline kinase-α (Chk) and phosphatidylcholine specific phospholipase D (PC-PLD) have been associated with increased malignancy [1, 3]. Chk is a cytosolic enzyme that is responsible for phosphorylation and converting free choline to phosphocholine. Two mammalian isoforms of PC-PLD, PLD1 and PLD2 have currently been identified. While PLD2 is restricted to cytoplasm, PLD1 shuttles between cytoplasm and membrane upon activation by G proteins such as ARF, Rho and Rac [4]. Here we used small interfering RNA (siRNA) against Chk and PLD1 alone or in combination in triple negative MDA-MB-231 human breast cancer cells to silence these two genes. Effective downregulation was confirmed at both protein and mRNA levels. However, downregulation of Chk with siRNA resulted in increased PLD1 expression, and downregulation of PLD1 resulted in increased Chk expression, typifying the ability of cancer cells to adapt. We sought to further understand mechanisms underlying this interdependence by changing the concentrations of free choline in the media. We observed a reduction in the increase of PLD1 protein expression and mRNA levels following Chk downregulation with higher concentration of free choline (107.1µM), suggesting that free choline plays a role in mediating the increase of PLD1 observed following Chk downregulation. Understanding the complex networks of pathways that participate following downregulation of a specific target is critical in a complex disease like cancer. These participating pathways provide novel opportunities to target in concert with the primary target to achieve improved control and minimize compensatory responses that allow cancer cells to survive or adapt. This is especially important if the downregulation of one ‘oncogenic target’, e.g. Chk, results in the upregulation of another, e.g. PLD1.

Acknowledgements: This work was supported by NIH P50 CA103175.

References: 1. Glunde, K. et al., Expert Rev Mol Diagn, 2006; 2. Bañez-Coronel M. et al., Curr Cancer Drug Targets, 2008; 3. Toschi, A. et al., Cancer Letters, 2010; 4. Huang, P. and Frohman, MA., Expert Opin Therapy, 2007.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1255. doi:10.1158/1538-7445.AM2011-1255
Abstract 1258: Regulation of energy metabolism and protein synthesis by the Pim protein kinases
Authors: Zemskova Marina Yu , Beharry Zanna , Mahajan Sandeep , Kraft Andrew S.
Published: June 2012

The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked mTORC1 activity by stimulating the phosphorylation, and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases (TKO MEFs) demonstrated activated AMPK driven by elevated ratios of AMP:ATP relative to wild type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5′-cap dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and PGC-1α, enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. These results demonstrate the Pim kinase -mediated control of energy metabolism through regulation of AMPK activity, and identify a new and important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth. In prostate cancer cell lines we find that the Pim-3 protein kinase is elevated in those cells that are highly metastatic including PC3-LN4, ARCaPM and M12 when compared to PC-3, ARCaPE and p69 and the amount of Pim-3 correlates with c-Myc levels. Further experiments will be needed to determine whether the Pim-3 protein kinase expression regulates the metastatic potential of prostate cancer cells through control of energy metabolism and protein synthesis.

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 1258. doi:10.1158/1538-7445.AM2011-1258
Abstract 1259: c-Myc influences mitochondrial structure and function by regulating fusion and fission
Authors: Graves J. Anthony , Rothermund Kristi D. , Wang Yudong , Elster Jennifer , Vockley Jerry , Van Houten Bennett , Prochownik Edward V.
Published: June 2012

Background: c-Myc (Myc) is a global transcription factor that controls the expression of a large part of the human genome. The numerous Myc target genes function in a variety of normal cellular processes whose collective expression of these processes is referred to as the “Myc phenotype”. One such cellular process is the regulation of mitochondrial biogenesis. Objectives: To understand the specific influence that Myc has on mitochondrial structure and function. We hypothesize that Myc is involved in the regulation of both the production and destruction of mitochondria, and that the ability of Myc to maintain this balance has an impact on cellular transformation. Design/Method: Various cell lines that allow for the manipulation of Myc levels have been used. These include cells that have been engineered to have inducible Myc over-expression as well as inducible expression of Myc shRNA. These strains were used in a series of assays that measured different aspects of the Myc effect on mitochondrial structure and function. Most notably, metabolic pathways were measured by an extracellular flux analyzer and electron and confocal microscopy was performed to examine the structure of the mitochondria in detail. Results: The induction of Myc results in dramatic increases in overall mitochondrial mass, as well as increasing individual mitochondrial size and cristae density. This increase in recognizable mitochondria correlates to increases in oxidative phosphorylation and decreased mitochondrial loss through mitophagy. Finally, Myc levels correlate to increased mitochondrial inter-connectivity. This appears to be related to the ability of Myc to influence the expression of the mitochondrial fusion protein, Opa1 and the fission protein, DLP1. Conclusions Reached: The changes in mitochondrial structure and function are directly influenced by the expression of Myc. There appear to be multiple pathways involved in maintaining a specific mitochondrial mass, which emphasizes the importance of this organelle in cellular homeostasis.

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 1259. doi:10.1158/1538-7445.AM2011-1259
Abstract 1272: Role of c-Myc, Oct1 and KLF4 in cell metabolism: Involvement of pluripotency factors in metabolic remodeling during tumorigenesis
Authors: Bellance Nadege , Karedath Thasni , Caneba Christine , Pabst Lisa , Asad Syed , Nagrath Deepak
Published: June 2012

Embryonic stem cells have the ability to give rise to different cell types and also the self reniewal capacity. Those features are essentially controlled by pluripotency factors which maintain cells into an undifferentiated state. Cancer cells are distinguished from normal cells by a dedifferentiation process in which the expression of pluripotency factors can be increased. c-Myc, Oct4 and KLF4 are pluripotency transcription factors. Oct-1, homologue of Oct-4, have been recently revealed to be pro-tumorigenic and his expression, such as c-Myc, is increased in various cancers. In this study, we investigated the role of this transcription factors into the cell metabolism. The role of the oncogene MYC on tumor cell metabolism has been largely studied. In cancer cells, c-Myc induces the expression of genes involved in glycolysis and glutaminolysis. However, the entire metabolic changes induced by c-Myc have not yet been elucidated. Furthermore, little is known about the role of Oct-1 and KLF4 in cancer metabolism. We have used the metabolic footprinting approach in order to characterize the Oct-1, c-Myc and KLF4 deficient cells and to identify the induced consecutive metabolic remodeling. Because of the variability of mutations observed in cancer cells, we investigated the effects induced by Oct-1, KLF4 and c-Myc deficiencies in mouse and rat embryonic fibroblasts by using UPLC (Ultra performance liquid chromatography). The uptake and release of metabolites was quantified by providing different substrates (glutamine or fatty acid). The glucose consumption and the lactate production which defined as the Warburg effect was measured. The mitochondrial function was investigated by oxygen consumption rate measurement. We found that the metabolites produced by Oct-1, KLF4 and c-Myc deficient cells seems to present a signature which can explain there over-expression in tumors. Our results suggest that embryonic stem cells related pluripotency transcription factors are implicated into the metabolic remodeling of cancer cells.

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 1272. doi:10.1158/1538-7445.AM2011-1272
Abstract 1285: Genetic variations predicting cisplatin cytotoxicity associated with the overall survival in lung cancer patients receiving platinum-based chemotherapy
Authors: Tan Xiang-Lin , Moyer Ann M. , Schaid Daniel J. , Niu Nifang , Batzler Anthony , Li Liang , Sun Zhifu , Yang Ping , Wang Liewei
Published: June 2012

Background: Candidate gene and candidate pathway-based pharmacogenomic studies have been used to investigate the inherited variability of prognosis of lung cancer treated with platinum-based chemoptherapy. In order to make it possible to conduct an ‘unbiased’ query across the entire genome, a genome-wide association study (GWAS) in 283 lymphoblastoid cell lines (LCLs) was carried out to help identify candidate SNPs associated with variation in cisplatin cytotoxicity. Furthermore, we set out to test the hypothesis that genetic variation resulting in differences in cisplatin cytotoxicity might influence the overall survival of patients with lung cancer treated with platinum-based antineoplastic agents.

Material and Methods: We applied ethnically diverse Coriell “Human Variation Panel” LCLs for GWAS. Then, we picked 168 top significant SNPs selected from GWAS in 283 LCLs and genotyped these SNPs in 1183 lung cancer patients (222 SCLC and 961 NSCLC) who received platinum-based chemotherapy. Genotyping was performed using the Illumina Golden Gate platform. The association of SNPs with lung cancer overall survival was analyzed by Cox regression model after adjusting for disease stage. Functional validation of candidate genes was performed by siRNA screening in NSCLC cell lines CRL5872, H1299 and SCLC cell line CRL5823.

Results: A total of 157 SNPs were successfully genotyped for 1183 lung cancer patient samples. The Cox regression analysis indicated that 9 and 10 different SNPs were associated with overall survival for NSCLC and SCLC patients, respectively (p value <0.05). Fifteen genes including 7 genes located in the region of 200kb up or downstream of 4 SNPs (rs11169748, rs2440915, rs5952066 and rs7620841) and 8 genes for which the expression was correlated with three SNPs (rs11169748, rs2440915 and rs5952066) were selected for siRNA screening. Knockdown of DAPK3 and METTL6, for which the expression levels were correlated with the SNPs rs11169748 and rs2440915, significantly decreased cisplatin sensitivity in lung cancer cell lines CRL5872, H1299 and CRL5823.

Conclusions: This series of clinical and complementary laboratory-based functional studies identified several candidate genes and SNPs that might help predict treatment outcomes of platinum-based therapy in lung cancer.

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 1285. doi:10.1158/1538-7445.AM2011-1285