bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-116: sLZIP induces migration and invasion of cervical cancer cells through expression of matrix metalloproteinase-9
Authors: Kang Hyereen , Jang Sung-Wuk , Ko Jesang
Published: June 2012

Extracellular proteolysis mediates tissue homeostasis. In cancer, altered proteolysis leads to abnormal tumor growth, inflammation, tissue invasion, and metastasis. Chronic infections and inflammation are major risk for various types of cancer. The matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with inflammation and tumorigenesis. Matrix metalloproteinase-9 (MMP-9) is involved in a wide range of normal and pathologic conditions, including inflammation, tissue repair, tumor invasion, and metastasis. sLZIP is a member of the zinc-finger transcription factor family. However, role of sLZIP in MMPs expression is previously unknown. Our results showed that sLZIP increased the mRNA level and the proteolytic activity of MMP-9 in HeLa cells. sLZIP also increased the transcription activity of MMP-9 by binding to the CRE of the MMP-9 promoter region. The requirement for sLZIP in MMP-9 expression was further supported by the fact that HeLa cells expressing sLZIP siRNA was refractory to MMP-9 expression. Results from wound healing and invasion assays showed that sLZIP enhanced migration and invasion of cervical cancer cells. Increased migration and invasion of HeLa cells induced by sLZIP was restored by inhibition of the proteolytic activity of MMP-9. Our results indicate that sLZIP induces expression of MMP-9 and is probably involved in invasion and metastasis of cervical 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 LB-116. doi:10.1158/1538-7445.AM2011-LB-116
Abstract LB-122: Single copy detection of KRAS mutations in colorectal cancer tissue and blood by qPCR using primers with enhanced analytical selectivity
Authors: Chupreta Sergey , Kelchner Vanessa , Kurihara Laurie , Makarov Vladimir
Published: June 2012

The development of highly sensitive genotyping assays that are suitable for clinical diagnostics opens new opportunities for the detection, assessment and management of cancer. Anticipated uses for these assays include profiling tissue biopsies and circulating tumor cells (CTCs), and detecting mutations in circulating cell-free nucleic acids. Swift Biosciences has developed myT™ Primers which have unique thermodynamic properties that make them highly sensitive to mismatch discrimination. myT™ Primer qPCR assays have been developed for the seven most frequent KRAS mutations in codons 12 and 13. These assays demonstrated single copy sensitivity for all seven KRAS mutations when used to genotype formalin fixed paraffin embedded (FFPE) specimens from Stage III colorectal tumors. When compared to a leading commercially available KRAS qPCR mutation test kit, myT™ Primer KRAS assays demonstrated several orders of improved specificity. A myT™ Primer assay was also developed for BRAF V600E/K mutations; results from FFPE melanoma specimens indicate high sensitivity and specificity. As a proof of concept for CTC detection, limiting dilutions of cells harboring the KRAS G12V mutation were added to whole human blood. Single copy detection of G12V was observed from an equivalent of a few microliters of blood. Because CTCs and cell-free cancer DNA can exist in blood at extremely low frequencies, we tested assay performance at even lower target concentrations. Mutant KRAS plasmid templates were titrated with wt KRAS plasmids such that the total KRAS copy number was 1×108. Under these conditions, the G12V-specific assay was able to detect the presence of ten mutant templates per 100 million (108) wt templates. The extreme selectivity of the myT™ Primer assays may make them especially useful for detection of mutations present at extremely low copy number and for genotyping difficult samples such as needle biopsies, CTCs and serum.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-122. doi:10.1158/1538-7445.AM2011-LB-122
Abstract LB-13: Calcitonin receptor-zonnula occludens-1 interaction is critical for calcitonin-induced destabilization of tight junctions and s prostate cancer metastasis
Authors: Shah Girish V. , Tamrakar Pratistha , Thomas Shibu , Lakshmikanthan Vijaybasker , Parajuli Parash , Sathyanarayanajois Seetharama , Bahouth Suleiman , Fanning Alan , Anderson James
Published: June 2012

Most primary prostate cancers (PCs) and PC cell lines express calcitonin (CT) and/or its receptor (CTR), and their co-expression positively correlates with tumor grade of primary PCs and growth/invasiveness of PC cell lines. Activation of the CT-CTR axis in non-invasive PC cells induces an invasive phenotype. In contrast, silencing of CT-CTR axis markedly reduces the tumorigenicity and metastatic capacity.

We have identified that cytoplasmic (C) tail of CTR contains a canonical class I type PSD-95, Discs-large, Zonnula Occludens-1 (PDZ)-binding motif, mutation of which does not affect its ability to bind its ligand or activate its G protein-coupled actions but abrogates CT-elicited destabilization of tight junctions or invasion. We then used CTR-C tail as a bait to identify its interactor by yeast two hybrid screening of prostate cDNA library. This revealed that CTR-C tail binds to intracellular membrane protein zonula occludens (ZO-1) through its PDZ domain. This interaction was confirmed using multiple approaches such as pull-down assays, overlay assays, co-immunoprecipitation and acceptor photobleaching FRET microscopy. The results demonstrate that CTR binds to PDZ3 domain of ZO-1 through its PDZ-binding motif. Mutation of either CTR-C PDZ-binding motif or ZO-1- PDZ3 domain abrogated actions of CT on tight junctions, invasion, orthotopic tumor growth and promotion of distant metastasis.

These results for the first time demonstrate PDZ-dependent interaction between CTR and ZO-1; and that this interaction is critical for CT-CTR axis-elicited prostate cancer progression. Considering overexpression of CT-CTR axis in malignant prostate, the results suggest that prevention of this interaction may serve as a new therapeutic possibility for the treatment of advanced prostate 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 LB-13. doi:10.1158/1538-7445.AM2011-LB-13
Abstract LB-137: YAP promotes breast cell proliferation and survival partially through stabilizing the KLF5 transcription factor
Authors: Zhi Xu , Zhao Dong , Zhou Zhongmei , Liu Rong , Chen Ceshi
Published: June 2012

The Yes-associated protein (YAP) is an oncoprotein in the Hippo tumor suppressor pathway that regulates tumorigenesis. Although YAP functions through its WW domains, the YAP WW domain binding partners have not been completely determined. In this study, we demonstrate that YAP functions partially through binding to KLF5, a transcription factor promoting breast cell proliferation and survival. YAP interacts with the KLF5 PY motif through its WW domains, prevents WWP1, a KLF5 E3 ubiquitin ligase, from binding to KLF5, and stabilizes KLF5 by decreasing the WWP1-mediated KLF5 ubiquitination and degradation. Over-expression of the wild type but not WW domain deficient YAP up-regulates the KLF5 protein levels and the mRNA expression levels of the KLF5 downstream target genes, including FGF-BP and ITGB2. In addition, knockdown of YAP decreases the expression levels of KLF5, FGF-BP, and ITGB2. Depletion of either YAP or KLF5 decreases breast cell proliferation and survival in MCF10A and SW527 breast cells. Importantly, stable knockdown of YAP or KLF5 significantly suppresses SW527 xenograft growth in mice. The YAP upstream kinase LATS1 suppresses the KLF5-FGF-BP signaling axis and cell growth through YAP. Finally, both YAP and KLF5 are co-expressed in estrogen receptor (ERα)-negative breast cell lines. These findings suggest that KLF5 is an important transcription factor partner for YAP and contributes to the Hippo tumor suppressor pathway. This study is supported by a grant from the American Cancer Society (RSG-08–199–01) and a grant from the Department of Defense (W81XWH-07–1–0191).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-137. doi:10.1158/1538-7445.AM2011-LB-137
Abstract LB-139: A novel mechanism of tumor suppression by Piasy through NF-kappaB/RelA DNA binding repression and cytoplasmic translocation
Authors: Bridges Rebecca , Wang Yun , Wortham Aaron , Swanzey Emily , Stupor Zach , Lagowski James , Kulesz-Martin Molly , Liu Yuangang
Published: June 2012

NF-kappaB activation is commonly associated with malignancy. Constitutive NF-kappaB activation contributes to tumor growth, metastasis, and resistance to chemotherapy and radiation therapy. Activators of NF-kappaB in human cancer include viral oncogenes, inflammatory cytokines, and the loss of tumor suppressors like CYLD. Loss of the protein inhibitor of signal transducer and activator of transcription family member Piasy is associated with malignant progression in chronic myelogenous leukemia and myelodysplastic syndrome, suggesting its role as a tumor suppressor. In this study, we provide evidence for a novel mechanism for NF-kappaB activation in human cancer through deficiency of Piasy. Piasy repressed NF-kappaB luciferase activity in response to TNFalpha, constitutively active IKK2, or overexpressed RelA. Piasy protein directly bound to RelA, through the C terminus of Piasy and the DNA binding domain of RelA, distinguishing it from other Pias family proteins that bind to RelA. Piasy inhibited DNA binding of RelA in cells, as detected by DNA affinity immunoblotting and chromatin immunoprecipitation. However, the DNA binding activity of purified RelA protein was not affected by purified Piasy protein, suggesting that Piasy indirectly represses RelA DNA binding in vivo. Analysis of cellular localization of Piasy and RelA proteins revealed that Piasy colocalized with RelA in the cytoplasm, correlated with enhanced RelA cytoplasmic translocation. In vivo consequences of Piasy depletion were shown in a mouse model of psoriasis-like epidermal hyperplasia in mice, and in human squamous cell carcinomas (SCC) of the skin. Piasy null mice topically treated with NF-kappaB activator imiquimod exhibited increased nuclear RelA in epidermis compared to wild type controls. Piasy levels in human skin SCC were inversely associated with nuclear RelA. These findings suggest a novel mechanism for Piasy as a tumor suppressor by the inhibition of NF-kappaB oncogenic pathways in human cancers. (This work was supported by NIH 5R01CA098577, and 5R01AR55651)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2011-LB-139
Abstract LB-140: Menin-mediated regulation of pancreatic β-cell differentiation factors
Authors: Shi Kerong , Parekh Vaishali , Roy Swarnava , Agarwal Sunita K.
Published: June 2012

Pancreatic endocrine tumors occur sporadically or in familial multiple tumor syndromes such as MEN1 (Multiple Endocrine Neoplasia type 1). Biallelic loss/inactivation of the MEN1 tumor suppressor gene is only found in 20–30% of sporadic pancreatic endocrine tumors. Therefore, identification of other molecular and genetic causes of pancreatic endocrine tumorigenesis is an area of intense investigation. Because MEN1 is the main gene reported so far that affects pancreatic endocrine tumors, analyzing downstream effects of menin (MEN1 gene encoded protein) could uncover other candidate genes and pathways responsible for pancreatic endocrine tumorigenesis. During the process of endocrine pancreas development, a cascade of transcription factors under tight sequential- and temporal-regulated expression ensures normal organogenesis. Alteration of such developmental and differentiation factors could potentially lead to tumorigenesis.

To study cellular transformation related to menin loss in the pancreatic β-cell, we used the mouse pancreatic β-cell line MIN6 as a model system. RNA and protein were isolated from MIN6 cells after menin knockdown or after menin over-expression as well as from their respective controls. Twelve transcription factors associated with β-cell differentiation were analyzed using QRT-PCR and western blot: Hlxb9, Hnf3β, Isl-1, MafA, MafB, NeuroD1, Ngn3, Nkx2.2, Nkx6.1, Pax4, Pax6, and Pdx1.

Three different sources of menin siRNAs and four different transfection reagents were tested in time course knockdown experiments. Significant menin knockdown in MIN6 cells was observed with 74.3% ± 15.03% (n=6) knockdown efficiency at 72 hours post-transfection. For transient menin over-expression in MIN6 cells, the transfection efficiency was close to 60% (based on GFP plasmid transfection). Endogenous expression of all factors, except Ngn3, was detectable in MIN6 cells. None of the eleven transcription factors showed significantly altered RNA expression upon menin knockdown or upon menin over-expression. Western blot analyses showed 4–5 factors that were up-regulated upon menin knockdown, and down-regulated upon menin over-expression. Evolutionarily conserved promoter regions of Hlxb9, Isl-1, Nkx6.1, or Pax4 were not regulated upon menin knockdown or upon menin over-expression. These data indicate that menin could regulate some β-cell differentiation factors in a post-transcriptional manner. Mechanism of menin-mediated post-transcriptional regulation and downstream consequences on β-cell transformation and tumorigenesis is under investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-140. doi:10.1158/1538-7445.AM2011-LB-140
Abstract LB-142: Regulation of MYCN stability by reactive oxygen species in neuroblastoma
Authors: Ikegaki Naohiko , Regan Paul L. , Hicks Sakeenah L. , Maloney Nolan , Tang Xao X.
Published: June 2012

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Amplification of the MYCN proto-oncogene is associated with older age, rapid tumor progression, and the worst outcome of this disease. As MYCN amplification leads to its over-expression, high-level expression of MYCN is thought to cause an aggressive behavior of MYCN amplified tumors. In fact, the forced reduction of MYCN protein expression by siRNA results in growth suppression and apoptosis of NB cells with MYCN amplification. We have identified several compounds that can rapidly destabilize the MYCN protein (within a few hours), in NB cells and cause growth suppression. The compounds include FCCP, OSU-03012, and Salinomycin. Our recent data suggest that a common effect of the above compounds appears to be the inhibition of mitochondrial functions.

Moreover, ascorbic acid, an anti-oxidant, abolishes the effect of these compounds on MYCN protein stability. It is known that inhibition of mitochondrial oxidative phosphorylation increases the production of reactive oxygen species (ROS), which include superoxide (O2-), hydroxyl radical (•OH), and hydrogen peroxide (H2O2). Anti-oxidants are also known to quench ROS. Preliminary data showed that ROS was detected when the IMR5 NB cells were treated with OSU-03012 for three hours. In addition, we have found that forced over-expression of pVHL, an E3 ubiquitin ligase, in NB cell lines results in reduction of MYCN protein levels. We are currently testing whether ROS regulates MYCN stability by hydroxylation of proline residues on MYCN, and whether pVHL recognizes the ROS-modified MYCN, which is then subjected to rapid proteasome-dependent degradation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-142. doi:10.1158/1538-7445.AM2011-LB-142
Abstract LB-213: Acheron augments triple negative breast cancer stem-like cells and exacerbates chemotherapeutic and radiation resistance
Authors: Scully Steve J. , Compton Shannon , Francescone Ralph A. , Schwartz Lawrence , Yan Wei , Shao Rong
Published: June 2012

Basal-like breast cancers that are negative for clinically important receptors (estrogen, progesterone, and Her2/neu), carry the worst prognosis for patients that are diagnosed with breast cancer. Acheron is a novel protein that was recently demonstrated to be highly expressed in triple-negative basal-like breast cancers. Previous work in our lab has demonstrated that breast cancer cell lines that are engineered to express ectopic Acheron are more invasive, have higher levels of MMP-9 and VEGF, and develop increased tumor burden in vivo in xenografted SCID/beige mice.

In order to better understand the role Acheron may play in these basal-like breast cancers, the triple negative breast cancer cell lines MDA 231 and MDA 435 were engineered to express ectopic Acheron. Engineered cells were cultured in serum free media with EGF and FGF to cultivate them as mammary cancer stem-like cells. Relative to empty vector control cells, Acheron-expressing MDA 435 cells demonstrated a greater ability to form acini-like structures in suspension as well as support anchorage independent growth.

Cells that were cultivated in basal media with serum were also analyzed for cell adhesion and expression of the laminin adhesion protein CD29 (Beta-I Integrin). Expression of Acheron in either MDA 231 and 435 cells lead to a marked reduction in substrate adhesion on laminin-coated plates relative to controls. Consistent with this observation, Acheron-expressing cells also expressed reduced levels of CD29.

FACS analysis for the apoptosis markers Annexin-V binding and propidium iodide exclusion demonstrated that Acheron-expressing cells were 2–3 times less sensitive to the same concentration of the chemotherapeutic drugs: Doxorubicin and docetaxel, and to a lesser extent to ionizing radiation (1.6 fold).

In summary, we have extended the analysis of Acheron action in triple-negative basal-like breast cancer cells. We have found that these cells that express ectopic Acheron are more resistant to chemotherapeutic drugs and radiation and have a greater capacity to grow in an anchorage-independent manner. These data support the hypothesis that Acheron expression in triple-negative breast cancers may confer properties that would be anticipated to result in a poorer prognosis. Therefore, Acheron deserves attention and further investigation as a new cancer target in triple-negative basal-like breast 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 LB-213. doi:10.1158/1538-7445.AM2011-LB-213
Abstract LB-220: TMEM16A, a novel calcium-activated chloride channel, modulates tumor proliferation via MAPK and Cyclin-D1 signaling
Authors: Shiwarski Daniel , Bertrand Carol , Egloff Ann Marie , Huang Xin , Seethala Raja , Grandis Jennifer , Gollin Susanne , Duvvuri Umamaheswar
Published: June 2012

TMEM16A, a calcium-activated chloride channel, was recently isolated from oral cavity cancer cells. TMEM16A is amplified in head and neck cancers and overexpressed in gastrointestinal stromal tumors (GIST's); however, the role that TMEM16A plays in promoting tumor growth remains unclear. The goal of this research was to establish the biologic function of TMEM16A in the context of epithelial malignancies. We hypothesized that TMEM16A contributes to tumor cell survival and may be a useful prognostic factor for patients with squamous cell carcinoma of the head and neck (SCCHN). Knockdown and overexpressing SCCHN and T24 cell lines were transduced with lentiviral particles encoding RNAi and engineered to stably overexpress TMEM16A. For functional verification, electrophysiologic assays were performed to establish that the altered TMEM16A expression was modulating the chloride current. To elucidate a mechanism of TMEM16A's proliferative effects, immunoblotting and pharmacologic inhibition of potential downstream targets were performed. Our results demonstrate that the levels of TMEM16A expression modulates proliferation in vitro and in vivo, and that stable overexpression of TMEM16A increases resistance to cisplatin therapy as compared to cancer cell lines expressing endogenous TMEM16A. Specificity of these phenotypic results were confirmed using genetic rescue experiments. Next, we identified that the MAPK/Cyclin-D1 axis was a critical signaling pathway in TMEM16A-induced proliferation, and that pMAPK/MAPK and Cyclin-D1 levels paralleled expression of TMEM16A. Finally, using pharmacologic agents niflumic acid or NPPB to inhibit the TMEM16A chloride channel activity in combination with a MAPK inhibitor (U0126) demonstrated additive effects significantly decreasing cancer cell proliferation in vitro. In summary, our results provide novel insights into the biologic function of TMEM16A and its role in tumor cell proliferation. This research has enhanced our understanding about the pathogenesis of SCCHN tumors, and can potentially be translated to many other cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-220. doi:10.1158/1538-7445.AM2011-LB-220
Abstract LB-229: Arsenic trioxide, ascorbic acid and disulfiram (AAA) arrests pancreatic cancer development in a transgenic mouse model with p16 knockout and activated Kras
Authors: Dinnen Richard D. , Qiu Wanglong , Su Gloria H. , Fine Robert L.
Published: June 2012

Previously, we found that four human pancreatic cancer (PC) cell lines underwent robust cell death associated with dramatic ATP decline and ROS accumulation when treated with clinically achievable concentrations of arsenic trioxide, ascorbic acid and disulfiram (AAA). Early apoptosis was shifted to aponecrosis, and dependent on the voltage-dependent anion channel (VDAC), since shRNA directed against VDAC-1 abrogated the ROS accumulation and blocked cell death. In this study, assessment of AAA was performed in Pdx1-Cre transgenic mice with a conditional p16 knockout (p16flox/flox), and oncogenic KrasG12D. The KrasG12D, p16flox/flox; LSL-KrasG12D; Pdx1-Cre mice develop the full spectrum of pancreatic intraepithelial neoplasia (mPanIN) lesions, pancreatic ductal adenocarcinoma, and hepatic and peritoneal metastases. Over 95% of human PC have p16 deficiency and Kras mutation. Eight week old mice were treated for 8 weeks, 3 times/week with AAA, sacrificed, and the development of PC assessed. Little or no toxicity of the mice to the AAA treatment was observed. Frank PC was zero in AAA-treated mice and 31% in controls. The mean number of PanIN-3 lesions/mouse declined from 14.1 in controls to 1.58 in AAA-treated mice (p=0.008). The mean number of PanIN-2 lesions declined from 5.18 in controls to 1.67 in AAA treated mice (p=0.06). The mean number of PanIN-1 lesions in control mice were 14.2 and in AAA-treated mice were 16.0, p=0.51 indicating no difference in PanIN-1 occurrence. Preliminary results from immunohistochemical staining of the PanIN lesions for VDAC, indicated that PanIN-1 lesions have low, and PanIN-2 and 3 lesions have high, VDAC expression. This result parallels the in vitro human cell line data showing VDAC expression is critical to AAA sensitivity, and suggests a similar mechanism of action in vivo as in vitro. Although collection of data is ongoing, this is the first treatment that has been shown to arrest PanIN and tumor development in any a genetically-engineered mouse model for PC. The data suggests AAA either arrests the development of pre-cancerous lesions after the PanIN-1 stage, or the PanIN-2 and PanIN-3 lesions are destroyed by AAA in a VDAC-dependent manner.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2011-LB-229
Abstract LB-235: BCL6 enables Ph+ acute lymphoblastic leukemia cells to survive BCR-ABL1 kinase inhibition
Authors: Duy Cihangir , Hurtz Christian , Koeffler Phillip H. , Melnick Ari M. , Müschen Markus
Published: June 2012

Tyrosine kinase inhibitors (TKI) are widely used to treat patients with leukemia driven by BCR-ABL1 and other oncogenic tyrosine kinases. Recent efforts focused on the development of more potent TKI that also inhibit mutant tyrosine kinases. However, even effective TKI typically fail to eradicate leukemia-initiating cells, which often cause recurrence of leukemia after initially successful treatment. Here we report on the discovery of a novel mechanism of drug-resistance, which is based on protective feedback signaling of leukemia cells in response to TKI-treatment. We identified BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukemia-initiating subclones.

BCL6 is a known proto-oncogene that is often translocated in diffuse large B cell lymphoma (DLBCL). In response to TKI-treatment, BCR-ABL1 acute lymphoblastic leukemia (ALL) cells upregulate BCL6 protein levels by ∼90-fold, i.e. to similar levels as in DLBCL. Upregulation of BCL6 in response to TKI-treatment represents a novel defense mechanism, which enables leukemia cells to survive TKI-treatment: Previous work suggested that TKI-mediated cell death is largely p53-independent. Here we demonstrate that BCL6 upregulation upon TKI-treatment leads to transcriptional inactivation of the p53 pathway. BCL6-deficient leukemia cells fail to inactivate p53 and are particularly sensitive to TKI-treatment. BCL6-/-leukemia cells are poised to undergo cellular senescence and fail to initiate leukemia in serial transplant recipients. A combination of TKI-treatment and a novel BCL6 peptide inhibitor markedly increased survival of NOD/SCID mice xenografted with patient-derived BCR-ABL1 ALL cells. We propose that dual targeting of oncogenic tyrosine kinases and BCL6-dependent feedback represents a novel strategy to eradicate drug-resistant and leukemia-initiating subclones in tyrosine kinase-driven leukemia.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-235. doi:10.1158/1538-7445.AM2011-LB-235
Abstract LB-237: Epigenetic antagonism between Polycomb and SWI/SNF complexes duringoncogenic transformation
Authors: Wilson Boris G. , Wang Xi , Shen Xiaohua , McKenna Elizabeth S. , Lemieux Madeleine E. , Cho Yoon-Jae , Koelhoffer Edward C. , Pomeroy Scott L. , Orkin Stuart H. , Roberts Charles W.M
Published: June 2012

While epigenetic alterations are widely present in cancers, in the context of genomic instability it has been difficult to determine the degree to which epigenetic alterations serve as primary drivers of tumorigenesis. Cancers initiated by mutation of the SNF5 chromatin remodeling subunit, despite being highly aggressive and lethal, are diploid and genomically stable, making them an ideal model for this purpose. SNF5 is a core subunit of the SWI/SNF chromatin complex and is mutated in the large majority of malignant rhabdoid tumors (MRT), highly lethal cancers that occur in kidney, brain, and soft tissues of young children. Inactivating mutations in SNF5 have also recently been found to occur in a variety of other cancers and mutations in other SWI/SNF subunits occur frequently in ovarian, endometrial, renal, and lung cancers. Given our findings of genomic stability, we hypothesize that the consequences of SNF5 loss are fundamentally epigenetic in nature; that characterization of the epigenetic changes present in our model systems, rather than in cancers with genome instability, will identify those epigenetic alterations capable of driving oncogenic transformation; and lastly that cancers carrying mutations in the SWI/SNF complex can be effectively treated with epigenetically targeted therapies.

Analysis of Drosophila mutants suggests that Polycomb and SWI/SNF complexes can serve antagonistic developmental roles. However, the relevance of this relationship to human disease is unclear. Here we have investigated functional relationships between these epigenetic regulators in oncogenic transformation. Mechanistically, we show that loss of the SNF5 tumor suppressor leads to elevated expression of the Polycomb gene EZH2 and that Polycomb targets are broadly H3K27-trimethylated and repressed in SNF5-deficient fibroblasts and cancers. Further, we show antagonism between SNF5 and EZH2 in the regulation of stem cell-associated programs and that Snf5 loss activates those programs. Finally, using conditional mouse models, we show that inactivation of Ezh2 blocks tumor formation driven by Snf5 loss. Collectively, we demonstrate that the rapid onset of tumor formation following SNF5 loss arises due to imbalanced epigenetic antagonism between the SWI/SNF complex and the Polycomb complex PRC2. Our work reveals essential roles for epigenetic modifications during tumor formation and demonstrates that inactivation of EZH2 can have therapeutic efficacy against cancer in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-237. doi:10.1158/1538-7445.AM2011-LB-237
Abstract LB-247: Mutation and functional analysis of PIK3CA and PIK3R1 in endometrial cancer
Authors: Cheung Lydia Wai Ting , Hennessy Bryan T. , Li Jie , Yu Shuangxing , Myers Andrea P. , Djordjevic Bojana , Lu Yiling , Stemke-Hale Katherine , Zhang Fan , Ju Zhenlin , Cantley Lewis C. , Scherer Steve E. , Liang Han , Lu Karen H. , Russell Broaddus R. , Mills Gordon B.
Published: June 2012

The phosphatidylinositol 3 kinase (PI3K) pathway is the most frequently activated pathway across multiple tumor lineages and this is a potential therapeutic target. As a critical step to accelerate therapeutic development in endometrial cancer, we performed a comprehensive analysis of mutation and function of PI3K pathway members in a set of 243 highly characterized endometrial tumors. Whole gene resequencing revealed the highest frequencies of mutation in the PI3K pathway of any tumor lineage including PTEN (44%), PIK3CA (40%; encoding the p110α catalytic subunit of PI3K) and PIK3R1 (20%; encoding the p85a regulatory subunit). Indeed, when complete protein loss is considered, almost 80% of endometrioid endometrial cancers demonstrate an aberration in the PI3K pathway. Remarkably, mutations in the PI3K pathway were not mutually exclusive and indeed co-existence of PIK3CA or PIK3R1 mutation with heterozygous PTEN mutation occurred at frequencies higher than predicted by the frequency of each lesion alone. High-throughput reverse-phase protein array suggested that the dominant signaling effects occurred with PTEN protein loss and that PIK3CA or PIK3R1 mutation functionally mimic PTEN protein loss as indicated by protein markers including phosphorylated Akt and stathmin. Thus it appears likely that co-mutations in the PI3K pathway are selected to compensate haploinsufficiency of PTEN due to PTEN heterozygous mutation. To determine the functional consequences of mutations in PIK3R1, we demonstrated that several somatic PIK3R1 mutations tested conferred cytokine-independent growth on interleukin-3-dependent Ba/F3 cells and induced Akt phosphorylation in endometrial cancer cell lines indicating that they were gain of function mutations likely acting as oncogenes. Strikingly two of the gain of function mutations (E160* and R348*) lacked the ability to bind to the p110 catalytic subunit and E160* failed to bind to both p110 and PTEN. We demonstrate that E160* disrupts a novel mechanism of pathway regulation wherein p85 binds and stabilizes PTEN. Together, the data indicate that the PI3K pathway is targeted in the vast majority of endometrioid endometrial cancers representing a novel opportunity for implementation of targeted therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2011-LB-247
Abstract LB-25: Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling
Authors: Rubbi Liudmilla , Titz Björn , Brown Lauren , Glavan Erica , Komisopoulou Evangelia , Chen Sharon S. , Low Tracey , Tahmasian Martik , Skaggs Brian , Müschen Markus , Pellegrini Matteo , Graeber Thomas G.
Published: June 2012

In subtypes and late stages of leukemias driven by the tyrosine kinase fusion protein Bcr-Abl, signaling by the Src family kinases (SFKs) critically contributes to the leukemic phenotype. We performed global tyrosine phosphoprofiling using quantitative mass spectrometry of Bcr-Abl transformed cells in which the activities of the Src family kinases were perturbed to build a detailed context-dependent network of cancer signaling. Perturbation of the SFKs Lyn and Hck with genetics or inhibitors revealed Bcr-Abl downstream phosphorylation events either mediated by or independent of SFKs. We identified multiple negative feedback mechanisms within the network of signaling events affected by Bcr-Abl and SFKs, and found that Bcr-Abl attenuated these inhibitory mechanisms. The Csk binding protein Pag1 (also known as Cbp) and the tyrosine phosphatase Ptpn18 both mediated negative feedback to SFKs. We observed Bcr-Abl-mediated phosphorylation of the phosphatase Shp2 (Ptpn11) and this may contribute to the suppression of these negative feedback mechanisms to promote Bcr-Abl-activated SFK signaling. Csk and a kinase-deficient Csk mutant both produced similar globally repressive signaling consequences, suggesting a critical role for the adaptor protein function of Csk in its inhibition of Bcr-Abl and SFK signaling. The identified Bcr-Abl-activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-25. doi:10.1158/1538-7445.AM2011-LB-25
Abstract LB-251: The checkpoint molecules LAG-3 and PD-1 synergize to maintain tolerance to tumors
Authors: Goldberg Monica V. , Woo Seng-Ryong , Bankoti Jaishree , Selby Mark , Nirschl Christopher J. , Bettini Matthew L. , Vogel Peter , Grosso Joseph F. , Netto George , Chaux Alcides , Smeltzer Matthew , Workman Creg J. , Pardoll Drew M. , Korman Alan J. , Vignali Dario AA , Drake Charles G.
Published: June 2012

Checkpoint molecules like CTLA-4 are important regulators of tumor-specific tolerance, and monoclonal antibodies that block this class of molecules have emerged as important therapeutic modalities in multiple cancer types. Recent data suggest that tumor infiltrating, non-functional ("exhausted") CD8 cells may express more than one checkpoint molecule, implying a more subtle control system for modulating CD8 T cell function in vivo. Based on their frequent co-expression on tumor-infiltrating lymphocytes, we investigated the relative roles of PD-1 (programmed death - 1) and LAG-3 (lymphocyte activation gene -3) in tumor tolerance. Single knockout (KO) mice lacking either of these genes were relatively unaffected, with PD-1 KO mice developing a late-onset strain-specific myocarditis. However, double knockout mice (DKO) lacking both PD-1 and LAG-3 developed early autoimmune pathology, with infiltrating lymphocytes present in multiple organs, and eventual lethality. More significantly, DKO mice spontaneously rejected the poorly immunogenic B16 melanoma, while rejection was not observed when either PD-1 or LAG-3 was knocked out individually. Moreover, established tumors in wild-type mice could be cured by combined treatment anti-PD-1 and anti-LAG-3 antibodies, while single antibody treatment was sufficient only to delay tumor outgrowth. Taken together, these data demonstrate a previously unappreciated synergy between these two checkpoint molecules in tumor-specific tolerance, and suggest a novel combinatorial strategy for cancer immunotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-251. doi:10.1158/1538-7445.AM2011-LB-251
Abstract LB-257: Profiling the effects of IDH1 and IDH2 mutants on the glioma cell metabolome
Authors: Yan Hai , Reitman Zachary J. , Jin Genglin , Spasojevic Ivan , He Yiping , Bigner Darell D. , Karoly Edward D. , Yang Jian , Kinzler Kenneth W. , Vogelstein Bert
Published: June 2012

Somatic hotspot mutations in the NADP+-dependent isocitrate dehydrogenases, IDH1 and IDH2, arise frequently in gliomas. These gliomas include WHO grades II and III oligodendrogliomas and astrocytomas, as well as WHO grade IV secondary glioblastomas. When mutated, IDH1 and IDH2 gain the ability to produce (R)-2-hydroxyglutarate (2HG) and may dominant-negatively inhibit IDH1-WT. Aside from these specific functions, the downstream effect of these metabolic enzyme mutations on cellular metabolism is unknown. To identify metabolic alterations caused by IDH1 and IDH2 mutants, we used three mass spectrometry methods (GC-MS/MS, LC-MS/MS +ESI, and LC-MS/MS -ESI) to profile >200 biochemicals in lysates of human oligodendroglial cell line (HOG) cells that homologously express IDH1-R132H or IDH2-R172K, which are the most frequent IDH1 and IDH2 mutants in glioma, respectively. To determine whether 2HG production or inhibition of IDH1-WT could mediate similar effects on cellular metabolism as IDH mutant expression, we also analyzed cells treated cells with either 7.5mM or 30mM 2HG as well as cells with stable shRNA knockdown of endogenous IDH1-WT. Unsupervised hierarchical clustering, univariate threshold tests, correlation analysis, and principal component analysis revealed that cells expressing IDH1-R132H and IDH2-R172K have similar metabolite profiles, and that 2HG treatment also results in a similar profile. However, cell with shRNA IDH1 knockdown shared few metabolic features with cells expressing IDH mutants. Metabolite set enrichment analyses revealed that numerous amino acids, N-acetylated amino acids, TCA cycle metabolites, glutathione metabolites, and choline derivatives were markedly altered in IDH-mutant expressing cells. Strikingly, we found that N-acetyl-aspartyl-glutamate (NAAG), a common brain metabolite, is 50-fold reduced in cells expressing IDH1-R132H and >2-fold reduced in human glioma tissues with IDH1-R132H compared to glioma tissues without IDH mutations. This work shows that IDH mutants can induce widespread cellular metabolic changes. Additionally, the metabolites that were found to be altered in this study may play a role in glioma pathogenesis and may be useful for diagnosis or treatment for gliomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2011-LB-257
Abstract LB-258: Small-molecule inhibitors of de-ubiquitinating enzymes for cervical cancer treatment
Authors: Anchori Ravi , Sueblinvong Thanasak , Roden Richard , Bazzaro Martina
Published: June 2012

Title. Small-Molecule Inhibitors of de-Ubiquitinating Enzymes for Cervical Cancer Treatment Abstract: Human Papillomavirus (HPV) is the primary cause of cervical cancer and responsible for 5% of all cancers worldwide. While HPV vaccines can be an effective preventive measure against cervical cancer, there are currently no virus-specific therapies for it, and the efficacy of standard surgical and chemo/radiotherapies is limited for advanced disease. The E6 oncoprotein of HPV exerts its oncogenic activity by binding to the E3 ubiquitin ligase E6-AP and redirects its activity towards p53 and other tumor suppressor proteins for rapid ubiquitin-mediated proteasomal degradation. Therefore, stabilization of p53 via preventing its ubiquitin-mediated degradation represents a valid therapeutic approach for cervical cancer treatment. De-ubiquitinating enzymes are an attractive novel “druggable” target for cervical cancer as they are responsible for controlling the steady state levels of proteins (i.e. p53 and E6-AP) crucial for maintaining the transformed status of cervical cancer cells, upstream proteasome. Thus, their inhibition is potentially associated with less toxic effects as compared to inhibition of proteasomes.

During a structure-based screening of chalcone-based small-molecule inhibitors of ubiquitin-mediated protein degradation we have recently identified a novel class of molecules capable of inhibiting the ubiquitin-mediated protein degradation upstream proteasomes. The lead compound of the series, contains the α-β unsaturated carbonyl system, as the molecular determinant for inhibiting de-ubiquitinating enzyme activities and: i) is capable of inducing acute perturbation of the ubiquitin-mediated protein degradation pathways accompanied by accumulation of poly-ubiquitinated species and reduction of mono-, di-, and tri-ubiquitin species, ii) has no effect on the catalytic activities of purified proteasomes or proteasome in living cells, iii) is capable of induce aggresome formation, iv) is capable of rescuing p53 levels/functions in cervical cancer cells which are accompanied by decrease in the levels of Cyclin D1 and failure for the cells to enter the S-phase of the cell cycle, v) is capable of preventing tumor-colony formation and induces onset of apoptosis via caspase-3 activation in cervical cancer cells without affecting the viability of normal cells. Therefore, we believe in the feasibility of using our lead compound in preclinical model of cervical cancer to test its efficacy as antineoplastic agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2011-LB-258
Abstract LB-259: The gluconeogenic pathway is operative in lung cancer cells
Authors: Fan Teresa WM , Lorkiewicz Pawel , Higashi Richard M. , Lane Andrew N.
Published: June 2012

Accelerated aerobic glycolysis has been a hallmark of cancer metabolism since Otto Warburg's seminal discovery [1]. Glucose oxidation via glycolysis and the pentose phosphate pathway (PPP) not only produces energy and reducing equivalents but also anabolic precursors to meet the demands of proliferation and many essential functions of cancer cells. Although many of the anabolic precursors can be obtained from alternative fuel substrates, the de novo synthesis of ribose and therefore nucleotides normally requires glucose metabolism. Thus, under glucose deficiency, cancer cells will cease proliferation due to a lack of new synthesis of nucleic acids. This can occur in poorly vascularized areas of a tumor. We now have direct evidence that lung cancer cells can circumvent this problem by making ribose from Gln using part of the gluconeogenic pathway. By using U-13C5,15N2-Gln as tracer, we tracked the fate of the 13C and 15N atoms simultaneously into various metabolites of A549 and H1299 cells using FT-ICR-MS and NMR. We found that both labels were incorporated into ATP and other nucleotides. Although the expected 15N labeling patterns were found based on the purine biosynthetic pathway, the 13C labeling patterns show that the adenine ring and/or ribosyl unit of ATP were also labeled. This can only occur through the metabolic sequence of glutaminolysis, Krebs cycle, pyruvate carboxylation, PEP carboxykinase (PEPCK) reaction, gluconeogenesis, and PPP. We believe this is the first time that gluconeogenesis is demonstrated for cancer cell lines not known to be gluconeogenic, which could have important implications for how cancer cells adapt to a glucose-limited microenvironment and the role of Gln in such adaptations.

1. Warburg, O., On the origin of cancer cells. Science, 1956. 123: p. 309–314.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2011-LB-259