Human Tumor-derived Cell Research Articles

“The development tumor model” to study and monitor the entire progression of both primary and metastatic tumors

Glioblastoma multiforme and other malignant cancers resulting in solid tumors continue to be devastating diseases. In order to find more effective treatments, it is necessary to cultivate a better understanding of the dynamics of tumor development in relation to both primary and secondary tumors. Although hand-held or digital caliper methods can measure tumor growth in subcutaneous xenograft models, to date, the only way to follow and monitor the progression of growing tumors in orthotopic animal models is imaging. This is not enough. To improve our knowledge of the biological characteristics that take place during tumor progression at both primary and metastatic sites, it is indispensable to develop an in vivo model which enables us to reproduce, from the beginning to the end of the cancer's natural history, what really happens in a patient affected by a solid tumor. The ideal tumor model must allow us to monitor all the stages of the tumor's development, both in the primary bulk and in secondary locations, by obtaining cells, biopsies as well as performing stainings on sections. In this paper, "the development tumor model", already proposed by the author to monitor the whole progression of the glioblastoma, is also applied to the study of all solid malignancies. It is a xenogeneic orthotopic transplantation model using human tumor-derived cells from the pre-hypoxic phase as transplanted material, which will be cultured in a neurobasal serum-free medium. By transplanting the same material at the same time (time zero) into a number of immunodeficient and genetically identical mice or rats, the model can be used to create a pool of twin animal transplant candidates under the same testing conditions. By sacrificing one animal a week (or choosing other intervals as needed) and performing multiple biopsies and stainings on sections, we can monitor the entire development of both the primary and secondary tumors. This may shed light on which specific cells and particular markers need to be focused on in order to develop innovative, valid therapeutic strategies.

Triacetin‐based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma

Cancer is associated with epigenetic (i.e., histone hypoacetylation) and metabolic (i.e., aerobic glycolysis) alterations. Levels of N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate, are reduced in glioma; yet, few studies have investigated acetate as a potential therapeutic agent. This preclinical study sought to test the efficacy of the food additive Triacetin (glyceryl triacetate, GTA) as a novel therapy to increase acetate bioavailability in glioma cells. The growth-inhibitory effects of GTA, compared to the histone deacetylase inhibitor Vorinostat (SAHA), were assessed in established human glioma cell lines (HOG and Hs683 oligodendroglioma, U87 and U251 glioblastoma) and primary tumor-derived glioma stem-like cells (GSCs), relative to an oligodendrocyte progenitor line (Oli-Neu), normal astrocytes, and neural stem cells (NSCs) in vitro. GTA was also tested as a chemotherapeutic adjuvant with temozolomide (TMZ) in orthotopically grafted GSCs. GTA-induced cytostatic growth arrest in vitro comparable to Vorinostat, but, unlike Vorinostat, GTA did not alter astrocyte growth and promoted NSC expansion. GTA alone increased survival of mice engrafted with glioblastoma GSCs and potentiated TMZ to extend survival longer than TMZ alone. GTA was most effective on GSCs with a mesenchymal cell phenotype. Given that GTA has been chronically administered safely to infants with Canavan disease, a leukodystrophy due to ASPA mutation, GTA-mediated acetate supplementation may provide a novel, safe chemotherapeutic adjuvant to reduce the growth of glioma tumors, most notably the more rapidly proliferating, glycolytic and hypoacetylated mesenchymal glioma tumors.

Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue

Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissue obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer. Our study indicates that identifying gene expression and/or epigenetic differences between TdECs and NdECs may provide us with new anti-angiogenic targets. Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

FSH modulates the expression of inhibin‐alpha and the secretion of inhibins via orphan nuclear receptor NUR77 in ovarian granulosa cells

It has been previously reported that follicle-stimulating hormone (FSH) regulates the expression of inhibin-alpha in human granulosa cells, but the precise molecular pathway remains unknown. In the present study, we investigated the role of the orphan nuclear receptor, NUR77, in both the transcriptional regulation of the inhibin α-subunit gene and the secretion of inhibins. Our results showed that in a human granulosa cell tumor-derived cell line (KGN) and in human granulosa-lutein cells (hGL), FSH induced the expression of NUR77 and inhibin-alpha, although inhibin-alpha expression did not increased following FSH treatment if NUR77 was knocked down. Furthermore, simply overexpressing or reducing NUR77 levels affected inhibin-alpha expression, while NUR77 overexpression improved the secretion of inhibin A and B from human granulosa cells. In addition, chromatin immunoprecipitation-PCR, avidin-biotin-conjugated DNA precipitation, and luciferase reporter assays confirmed that NUR77 directly regulated the transcription of the inhibin-alpha gene through the specific NGFI-B response element located within its promoter. In the ovarian granulosa cells of the Nur77 knockout mice, the mRNA levels of inhibin-alpha were decreased relative to wild-type mice. These data indicate a role of NUR77 in the regulation of inhibin-alpha in ovarian granulosa cells.

Combined 5-FU and ChoKα Inhibitors as a New Alternative Therapy of Colorectal Cancer: Evidence in Human Tumor-Derived Cell Lines and Mouse Xenografts

BackgroundColorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.Methodology/Principal FindingsChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Conclusion/SignificanceOur data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Phenethylisothiocyanate alters site- and promoter-specific histone tail modifications in cancer cells.

Site-specific histone modifications are important epigenetic regulators of gene expression. As deregulation of genes often results in complex disorders, corrective modulation of site-specific histone marks could be a powerful therapeutic or disease-preventive strategy. However, such modulation by dietary compounds and the resulting impact on disease risk remain relatively unexplored. Here we examined phenethylisothiocyanate (PEITC), a common dietary compound derived from cruciferous vegetables with known chemopreventive properties under experimental conditions, as a possible modulator of histone modifications in human colon cancer cells. The present study reports novel, dynamic, site-specific chemical changes to histone H3 in a gene-promoter-specific manner, associated with PEITC exposure in human colon tumor-derived SW480 epithelial cells. In addition, PEITC attenuated cell proliferation in a concentration- and time-dependent manner, likely mediated by caspase-dependent apoptotic signalling. The effects of PEITC on histone modifications and gene expression changes were achieved at low, non-cytotoxic concentrations, in contrast to the higher concentrations necessary to halt cancer cell proliferation. Increased understanding of specific epigenetic alterations by dietary compounds may provide improved chemopreventive strategies for reducing the healthcare burden of cancer and other human diseases.

Abstract 4995: Tumor-dervied gammadelta regulatory T cells suppress innate and adaptive immunity through the induction of immunosenescence.

Abstract Fundamentally understanding the suppressive mechanisms utilized by different subsets of tumor-infiltrating regulatory T (Treg) cells is critical for the development of effective strategies for anti-tumor immunotherapy. Gammadelta Treg cells have recently been identified in human diseases including in cancer patients. However, the suppressive mechanisms and functional regulations of this new subset of unconventional Treg cells are largely unknown. In the current studies, we explored the suppressive mechanism(s) utilized by human breast tumor-derived gammadelta Treg cells on innate and adaptive immunity. We found that gammadelta Treg cells induced immunosenescence in targeted naïve and effector T cells, as well as dendritic cells (DCs). Furthermore, senescent T cells and DCs induced by gammadelta Treg cells had altered phenotypes and developed potent suppressive activities, further amplifying the immunosuppression mediated by gammadelta Treg cells. In addition, we demonstrated that manipulation of TLR8 signaling in gammadelta Treg cells can block gammadelta Treg-induced conversion of T cells and DCs into senescent cells in vitro and in vivo. Our studies identify the novel suppressive mechanism mediated by human tumor-derived gammadelta Treg cells on innate and adaptive immunity, which should be critical for development of strong and innovative approaches to reverse the tumor suppressive microenvironment and improve effects of immunotherapy. This work was supported by the grant from the American Cancer Society (RSG-10-160-01-LIB). Key words: gammadelta T cells; Regulatory T cells; Dendritic cells; Senescence; Immune suppression Citation Format: Jian Ye, Chunling Ma, Eddy C. Hsueh, Yanping Zhang, Mark A. Varvares, Daniel F. Hoft, Guangyong Peng. Tumor-dervied gammadelta regulatory T cells suppress innate and adaptive immunity through the induction of immunosenescence. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4995. doi:10.1158/1538-7445.AM2013-4995

Telomerase activity in human brain tumors: astrocytoma and meningioma.

Somatic cells do not have telomerase activity but immortalized cell lines and more than 85% of the cancer cells show telomerase activation to prevent the telomere from progressive shortening. The activation of this enzyme has been found in a variety of human tumors and tumor-derived cell lines, but only few studies on telomerase activity in human brain tumors have been reported. Here, we evaluated telomerase activity in different grades of human astrocytoma and meningioma brain tumors. In this study, assay for telomerase activity performed on 50 eligible cases consisted of 26 meningioma, 24 astrocytoma according to the standard protocols. In the brain tissues, telomerase activity was positive in 39 (65%) of 50 patients. One sample t test showed that the telomerase activity in meningioma and astrocytoma tumors was significantly positive entirely (P<0.001). Also, grade I of meningioma and low grades of astrocytoma (grades I and II) significantly showed telomerase activity. According to our results, we suggest that activation of telomerase is an event that starts mostly at low grades of brain including meningioma and astrocytoma tumors.

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

FOXO1 Controls Thyroid Cell Proliferation in Response to TSH and IGF-I and Is Involved in Thyroid Tumorigenesis

TSH and insulin/IGF-I synergistically induce the proliferation of thyroid cells mainly through the cAMP and phosphatidylinositol 3-kinase (PI3K) pathways. However, the events involved in this cooperative induction remain unknown, and molecules that are potentially controlled by both TSH and IGF-I are interesting candidates as integrators of both stimuli. The finding that the PI3K pathway is frequently activated in thyroid malignancies has attracted attention to this pathway in the thyroid field. One of the targets of PI3K is Forkhead box O (FoxO)-1, a widely expressed transcription factor involved in a variety of cellular processes such as differentiation, proliferation, and apoptosis. Here we show that FoxO1 is highly expressed in differentiated rat thyroid cells and human thyroid tissue compared with human thyroid tumor-derived cells and surgically removed thyroid tumors, in which its expression is reduced. In differentiated cells, TSH/cAMP treatment decreases FoxO1 mRNA and protein levels through proteasome activation, whereas both TSH and IGF-I control FoxO1 localization by promoting a rapid exclusion from the nucleus in an Akt-dependent manner. FoxO1 can control p27(KIP1) expression in differentiated and tumor cells of the thyroid. Furthermore, FoxO1 reexpression in tumor cells promotes a decrease in their proliferation rate, whereas FoxO1 interference in differentiated cells increases their proliferation. These data point to an important role of FoxO1 in mediating the effects of TSH and IGF-I on thyroid cell proliferation and provide a link between loss of FoxO1 expression and the uncontrolled proliferation of thyroid tumor cells.

Immuno-PET of the Hepatocyte Growth Factor Receptor Met Using the 1-Armed Antibody Onartuzumab

The overexpression and overactivation of hepatocyte growth factor receptor (Met) in various cancers has been linked to increased proliferation, progression to metastatic disease, and drug resistance. Developing a PET agent to assess Met expression would aid in the diagnosis and monitoring of responses to Met-targeted therapies. In these studies, onartuzumab, the experimental therapeutic 1-armed monoclonal antibody, was radiolabeled with (76)Br or (89)Zr and evaluated as an imaging agent in Met-expressing cell lines and mouse xenografts. (89)Zr-desferrioxamine (df)-onartuzumab was synthesized using a df-conjugate; (76)Br-onartuzumab was labeled directly. Met-binding studies were performed using the human tumor-derived cell lines MKN-45, SNU-16, and U87-MG, which have relatively high, moderate, and low levels of Met, respectively. Biodistribution and small-animal PET studies were performed in MKN-45 and U87-MG xenografts. (76)Br-onartuzumab and (89)Zr-df-onartuzumab exhibited specific, high-affinity Met binding (in the nanomolar range) that was concordant with established Met expression levels. In MKN-45 (gastric carcinoma) xenografts, both tracers cleared slowly from nontarget tissues, with the highest uptake in tumor, blood, kidneys, and lungs. (76)Br-onartuzumab MKN-45 tumor uptake remained relatively constant from 18 h (5 percentage injected dose per gram of tissue [%ID/g]) to 48 h (3 %ID/g) and exhibited tumor-to-muscle ratios ranging from 4:1 to 6:1. In contrast, (89)Zr-df-onartuzumab MKN-45 tumor uptake continued to accumulate from 18 h (10 %ID/g) to 120 h (23 %ID/g), attaining tumor-to-muscle ratios ranging from 20:1 to 27:1. MKN-45 tumors were easily visualized in imaging studies with both tracers at 18 h, but after 48 h (89)Zr-df-onartuzumab image quality improved, with at least 2-fold-greater tumor uptake than nontarget tissues. MKN-45 tumor uptake for both tracers correlated significantly with tumor mass and Met expression and was not affected by the presence of plasma shed Met. (89)Zr-df-onartuzumab and (76)Br-onartuzumab specifically targeted Met in vitro and in vivo; (89)Zr-df-onartuzumab achieved higher tumor uptake and tumor-to-muscle ratios than (76)Br-onartuzumab at later times, suggesting that (89)Zr-df-onartuzumab would be better suited to image Met for diagnostic and prognostic purposes.

Harnessing the power of human tumor-derived cell lines for the rational design of cancer therapies

In 2002, a mutated constitutively active form of the proto-oncogene BRAF kinase (BRAFV600E) was discovered in ~50% of patients with melanoma. Just last year, the FDA approved an inhibitor and a companion genetic test to treat metastatic melanomas that are positive for this mutation. The rational selection of drugs based on specific genetic variation(s) within a tumor represents the advent of personalized cancer therapeutics. Clearly, this is the way of the future but much work remains to be carried out – a fact that is also illustrated by the BRAFV600E case study. While regression of BRAFV600E tumors in a vast majority of metastatic melanoma patients treated with BRAF inhibitors is encouraging vis-a-vis efficacy of targeted cancer therapeutics, the response is transient and the tumors inevitably recur. In vitro studies have shown that resistance also arises when BRAF inhibitors are combined with therapeutics targeted to other members of the MAP kinase (MAPK) pathway. This can probably be attributed to the selection of resistant mutants from the extensive genetic diversity of tumors within a single patient with metastatic melanoma (Yancovitz et al., 2012). One potential strategy to prevent such recurrence is to hit multiple targets with a cocktail of drugs that are rationally selected to match the complete complement of oncogenic mutations within the heterogeneous tumor cell population. Rational design of such drug cocktails will undoubtedly benefit from understanding of the assorted mechanisms by which resistance develops: additional mutations, epigenetic changes, modification of drug metabolism or clearance, or switching to alternative pathways. However, for a targeted approach to be effective, it will ultimately have to be paired with technologies to comprehensively genotype the entire diversity of mutations within the tumor cell population (Baslan et al., 2012; Navin et al., 2011).

Interferon-β lipofection I. Increased efficacy of chemotherapeutic drugs on human tumor cells derived monolayers and spheroids

We evaluated the effect of hIFNβ gene transfer alone or in combination with different antineoplastic drugs commonly used in cancer treatment. Five human tumor-derived cell lines were cultured as monolayers and spheroids. Four cell lines (Ewing sarcomas EW7 and COH, melanoma M8 and mammary carcinoma MCF-7) were sensitive to hIFNβ gene lipofection. Although this effect appeared in both culture configurations, spheroids showed a relative multicellular resistance (insensitive colon carcinoma HT-29 excluded). EW7 and M8 hIFNβ-expressing cells were exposed to different concentrations of bleomycin, bortezomib, carboplatin, doxorubicin, etoposide, methotrexate, paclitaxel and vincristine in both configuration models. In chemotherapy-sensitive EW7 monolayers, the combination of hIFNβ gene and antineoplastic drugs displayed only additive or counteractive (methotrexate) effects, suggesting that cytotoxic mechanisms triggered by hIFNβ gene lipofection could be saturating the signaling pathways. Conversely, in chemotherapy-resistant EW7 spheroids or M8 cells, the combination of hIFNβ with drugs that mainly operate at the genotoxic level (doxorubicin, methotrexate and paclitaxel) presented only additive effects. However, drugs that also increase pro-oxidant species can complement the antitumor efficacy of the hIFNβ gene and clearly caused potentiated effects (bleomycin, bortezomib, carboplatin, etoposide and vincristine). The great bystander effect induced by hIFNβ gene lipofection could be among the main causes of its effectiveness, because only 1 or 2% of EW7 or M8 hIFNβ-expressing cells killed more than 60 or 80% of cell population, respectively.

Robust identification of transcriptional regulatory networks using a Gibbs sampler on outlier sum statistic

Identification of transcriptional regulatory networks (TRNs) is of significant importance in computational biology for cancer research, providing a critical building block to unravel disease pathways. However, existing methods for TRN identification suffer from the inclusion of excessive 'noise' in microarray data and false-positives in binding data, especially when applied to human tumor-derived cell line studies. More robust methods that can counteract the imperfection of data sources are therefore needed for reliable identification of TRNs in this context. In this article, we propose to establish a link between the quality of one target gene to represent its regulator and the uncertainty of its expression to represent other target genes. Specifically, an outlier sum statistic was used to measure the aggregated evidence for regulation events between target genes and their corresponding transcription factors. A Gibbs sampling method was then developed to estimate the marginal distribution of the outlier sum statistic, hence, to uncover underlying regulatory relationships. To evaluate the effectiveness of our proposed method, we compared its performance with that of an existing sampling-based method using both simulation data and yeast cell cycle data. The experimental results show that our method consistently outperforms the competing method in different settings of signal-to-noise ratio and network topology, indicating its robustness for biological applications. Finally, we applied our method to breast cancer cell line data and demonstrated its ability to extract biologically meaningful regulatory modules related to estrogen signaling and action in breast cancer. The Gibbs sampler MATLAB package is freely available at http://www.cbil.ece.vt.edu/software.htm. xuan@vt.edu Supplementary data are available at Bioinformatics online.

Mixed-1,10-phenanthroline–Cu(II) complexes: Synthesis, cytotoxic activity versus hematological and solid tumor cells and complex formation equilibria with glutathione

Cu(II) complexes with 1,10-orthophenanthroline (phen) show cytotoxic and antitumoral effects. To enhance and exploit these features, we studied complexes containing one or two phen units together with N,N′-substituted-imidazolidine-2-thione (L). We synthesized and structurally characterized the precursor molecule Cu(phen)(OH2)2(OClO3)2, and determined the complex formation constants of [Cu(phen)(L)]2+. We studied the cytotoxic activity of [Cu(phen)2(L)](ClO4)2 versus human hematologic (CCRF-CEM and CCRF-SB) and solid tumor-derived cell lines (K-MES-1, DU-145). The cytotoxic activities, in the 1–3μM range, show that our Cu(II)–complexes possess comparable inhibitory activities against both leukemia and carcinoma cells, unlike the majority of antineoplastic agents, usually more potent against hematologic cancer cells than against solid tumor cells. Because the free Cu(II) ion is reduced by glutathione (GSH), we studied the reactivity of our complexes with GSH, providing evidence that no redox reaction occurred under the chosen experimental conditions. Complex formation equilibria were present, studied by spectrophotometric titrations. The redox properties of the prepared compounds were also investigated by cyclic voltammetry, confirming that the mixed Cu(II) complexes were resistant to reduction.

Abstract 232: Protein kinase C b1 (PKCb1) inhibits the malignant phenotype of the human pancreatic tumor-derived cell line PANC1

Abstract Pancreatic cancer is a deadly disease with a 5% overall survival after five years. Different members of the PKC family have been described as modulators of pancreatic cancer progression but, till nowadays, the relevance of each PKC isoform in this process is unclear. The aim of the present work was to study the effect of PKCb1 overexpression, on several critical determinants of tumor progression using the human pancreatic tumor-derived cell line PANC1. Cells were stably transfected with an expression vector containing the full-length PKCb1 gene (PKCb1-PANC1) or with the empty vector, as control (pMTH-PANC1). While no morphologic alterations could be detected in vitro, PKCb1 overexpression induced a significant reduction in the proliferative capacity of PANC1 cells. The reduction in this capacity could be associated with a decrease in Cyclin D1, Cyclin E and phosphorylated ERK1/2 levels, and an increase in p21 expression levels, as measured by Western blot. In addition PKCb1-PANC1 cells showed an increased resistance to cell death induced by serum starvation (% viability: 58.54±4.1 vs 39.29±4.2 in pMTH-PANC1, p&amp;lt;0.05). Using flow cytometry we could determine that serum deprivation promoted PKCb1-PANC1 arrest in the G1 phase of the cell cycle (cells in G0/G1: 86.7±1.0% vs 45.5±0.8% in pMTH-PANC1, p&amp;lt;0.05). Moreover, the overexpression of PKCb1 reduced the in vitro invasive capacity using Matrigel coated transwells (cells x 10 microscopic x400 fields: 2.22±0.9 vs 10.10±2.6 in pMTH-PANC1, p&amp;lt;0.05). This behavior could be associated with a significant inhibition of secreted proteases activity (uPA: 4.11±0.8 vs 12.32±4.1 IU/mg/24h in pMTH-PANC1, p&amp;lt;0.05 and MMP-9: 15.24±0.03 vs 24.89±2.6 AU/mg in pMTH-PANC1, p&amp;lt;0.05). When control and PANC1-PKCb1 cells were s.c. inoculated into nude mice it was observed that PKCb1 cells were almost unable to grow and form tumors while vector-transfected cells retained this ability. In sum, our results suggest that PKCb1 overexpression in human pancreatic tumor cells PANC1 induced a less malignant phenotype both in vivo and in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 232. doi:1538-7445.AM2012-232

Abstract 5141: PI3K-mediated AMPK inactivation leads to a preneoplastic switch to glycolytic metabolism

Abstract Most proliferating and tumor cells generate the energy required to support rapid cell division by aerobic glycolysis instead of oxidative phosphorylation (Warburg effect). By extension, in order to meet the increased requirements of proliferation, they often display essential changes in energy metabolism and nutrient uptake pathways. Several oncogenes, including Myc HIF-1&amp;#945;, and AKT, drive this switch directly, by targeting Hexokinase II, Lactate Dehydrogenase A, Pyruvate Kinase M2, and Pyruvate Dehydrogenase Kinase 1. The AMP-activated protein kinase (AMPK) is a highly conserved protein kinase complex that plays a central role in the regulation of cellular energy homeostasis. The fact that AMPK is an evolutionarily conserved energy sensor and plays a role both downstream and upstream of defined tumor suppressors, suggests that it may function as a tumor suppressor; however, the exact contribution of AMPK to tumor progression is unclear. We have generated a Pten−/- mouse model in which the PI3K pathway is selectively activated in the thyroid epithelial cells, leading to hyperplasia which progresses to follicular carcinoma. The early hyperplastic lesions exhibit a dramatic down-regulation of the expression levels of many tricarboxylic acid cycle proteins due to reduced mRNA expression, which results in enhanced compensatory glycolysis (over 3-fold increase in lactate production in mutants vs. wt). In addition, the expression of most mitochondrially-encoded genes is heavily down-regulated, and none of the genes and proteins classically associated with the Warburg effect is found deregulated in the mutant thyroid cells. In this context, while AMPK is found to be activated in wt thyroids through phosphorylation on Thr172, AMPK is inactivated in mutant thyroids through PI3K-dependent phosphorylation on Ser485. Pten−/− mice treated with 5-aminoimidazole-4-carboxamide 1-&amp;#946;-D-ribofuranoside (AICAR, IP 500 mg/kg/day for 4 weeks) display a restoration of the metabolic and mitochondrially-encoded gene expression, as well as a significant decrease in the lactate production. Pharmacological PI3K inhibition in human tumor-derived thyroid cancer cell lines increases the expression levels of both TCA cycle and OXPHOS genes. Moreover, it also causes AMPK reactivation through Thr172 phosphorylation with a concomitant reduction in pAMPK Ser485. Based on these compelling data, we propose a novel mechanism leading to a Warburg-like effect in preneoplastic lesions: PI3K activation initiates through AMPK inactivation a coordinated rearrangement of the expression of metabolic genes, which favors aerobic glycolysis at the expense of TCA/OXPHOS. Further elucidation of this process will lead to innovative targeted approaches to selectively disrupt tumor progression, while preserving normal metabolism in non mutated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5141. doi:1538-7445.AM2012-5141

Abstract B47: Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control

Abstract The RB/E2F axis is frequently disrupted in multiple tumors. Classical studies reported that the RB tumor suppressor exerts its transcriptional repressor function through an interaction with the E2F family of transcription factors (E2F1-3) to control the timely expression of genes required for DNA replication and cell cycle progression. The E2F3 locus encodes two E2F3 isoforms, a and b, which have unique N-terminal sequences. Multiple studies indicate that E2F3, rather than E2F1 or 2, is elevated in various cancers, including prostate. In prostate tumors elevated E2F3 expression is an independent prognostic factor of clinical outcome, but the direct transcriptional targets of E2F3a and E2F3b have not been characterized. We found that E2F3a and b expression was increased in prostate tumor derived cell lines when compared to non-transformed controls. An expression array analysis following knockdown (k/d) of total E2F3 identified several unexpected targets including Interleukin 6 receptor (IL-6R), a critical component of the IL6 signaling cascade and calpain 2, a protease that is essential in cellular migration and has been shown to be elevated during prostate tumorigenesis. Further studies showed that the two genes were regulated by different E2F3 isoforms- IL6R was a target of E2F3a, while calpain 2 was regulated by E2F3b. Expression of both genes was enhanced by RB ablation. E2F3a-dependent IL-6R regulation was apparent in PC3, LNCaP, CWR-R1, and 22Rv1 cells. Chromatin immunoprecipitation (ChIP) studies identified sequences in the IL-6R promoter that were bound by E2F3. Transient co-transfection studies using an E2F3a expression plasmid showed that E2F3a transactivated the IL-6R promoter in a dose dependent manner. The IL-6R initiated signaling cascade was perturbed following a k/d of E2F3a since the levels of ERK1/2 phosphorylation was reduced. An siRNA-mediated ablation of each isoform revealed that E2F3b, not E2Fa, was effective is reducing calpain 2 levels. ChIP studies indicated that E2F3 binds to the endogenous calpain 2 promoter, and in transient transfection studies E2F3b transactivated the calpain 2 promoter in a dose dependent manner. An analysis of the calpain 2 promoter identified an androgen receptor (AR) half site. Additional transfection studies showed that the AR can cooperate with E2F3b in transactivation of the calpain 2 promoter. Moreover, reduced expression of E2F3b impaired cellular migration in a wound assay. The expression of the E2F3 isoforms has not been previously analyzed in the TRAMP prostate tumor model. Western immunoblot studies of five TRAMP tumors and three age and strain matched prostates showed that TRAMP tumors had highly elevated expression of both E2F3 isoforms. These results indicate that increased expression of both E2F3 isoforms is a feature of human tumor-derived cell lines and the TRAMP mouse model of prostate tumorigenesis. Calpain 2, expression was readily detected in all of the TRAMP tumors, but not in the control tissue. Hence E2F3 overexpression and increased calpain levels are features of human and mouse tumors. This analysis broadens the role of E2F3 in prostate tumorigenesis beyond the regulation of cell cycle progression. E2F3a is a link between the E2F/RB and the IL-6 signaling cascade, while E2F3b regulates expression of a protease that is essential in cell adhesion and migration. Therefore E2F3 deregulation affects multiple signaling networks to promote tumorigenesis. Citation Format: Stephen J. Libertini, Maria Mudryj, Alan P. Lombard, Honglin Chen, Veronica Rodriguez, Carlos Perez-Stable, Bushra al-Bataina, Tilak Koilvaram, Michael George, Allen C. Gao. Identification of novel E2F3 transcriptional targets expands the role of the RB/E2F axis beyond cell cycle control [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B47.

New Insights into p53 Signaling and Cancer Cell Response to DNA Damage: Implications for Cancer Therapy

Activation of the p53 signaling pathway by DNA-damaging agents was originally proposed to result either in cell cycle checkpoint activation to promote survival or in apoptotic cell death. This model provided the impetus for numerous studies focusing on the development of p53-based cancer therapies. According to recent evidence, however, most p53 wild-type human cell types respond to ionizing radiation by undergoing stress-induced premature senescence (SIPS) and not apoptosis. SIPS is a sustained growth-arrested state in which cells remain viable and secrete factors that may promote cancer growth and progression. The p21WAF1 (hereafter p21) protein has emerged as a key player in the p53 pathway. In addition to its well-studied role in cell cycle checkpoints, p21 regulates p53 and its upstream kinase (ATM), controls gene expression, suppresses apoptosis, and induces SIPS. Herein, we review these and related findings with human solid tumor-derived cell lines, report new data demonstrating dynamic behaviors of p53 and p21 in the DNA damage response, and examine the gain-of-function properties of cancer-associated p53 mutations. We point out obstacles in cancer-therapeutic strategies that are aimed at reactivating the wild-type p53 function and highlight some alternative approaches that target the apoptotic threshold in cancer cells with differing p53 status.