bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 994: Role of Akt isoforms in regulating intermediate filaments expression in cancer progression
Authors: Fortier Anne-Marie , Cadrin Monique , Asselin Eric
Published: February 2011

Keratins are epithelial-specific intermediate filament (IF) proteins, which are expressed in a tissue-specific manner. As part of the cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, a number of keratins are involved in intracellular signalling pathways which regulate the response to injuries and nonmechanical stresses, cell growth, cell death and cancer progression. Expression of keratins is maintained during malignant transformation of simple epithelial cells. However, vimentin, a mesenchymal-specific IF, is often co-expressed with keratins in late-stage cancer cells presenting a dedifferentiated phenotype. This is associated with an increased metastatic potential. Metastasis, the foremost cause of mortality in cancer patients, is a coordinated biological process involving multiple intracellular signalling pathways. PI3K/Akt pathway has been shown to play a role in invasion/metastasis. Moreover, the three Akt isoforms would play differential roles in this process. In the present study, we investigate the role of Akt isoforms in regulating the expression of keratins and vimentin in different epithelial carcinoma cell lines and the implication of IF in Akt-regulated cell invasion. Our results show that overexpression of constitutively active Akt isoforms up-regulates IF protein and mRNA levels, by increasing IF mRNA stability. On the contrary, down-regulation of Akt isoforms by shRNA decreases IF protein levels. To examine the regulation of IF expression under a more physiological condition, we treated cells with TGF-b1, which is known to induce Akt activation. We demonstrated that IF are up-regulated by TGF-b1 in PI3K-dependent manner. To our knowledge, these results represent the first indication that Akt activity is regulating IF expression. Overexpression of Akt isoforms also increases in vitro cell invasion. Interestingly, we showed that down-regulation of vimentin by siRNA decreases Akt2-induced cell invasion. These results support the hypothesis that modulation of IF expression plays an active role in cancer progression.

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 994.
Abstract 995: Integrin-Linked Kinase localizes in the primary cilia to mediate Hedgehog signaling
Authors: Barakat Badia , Vu Duc , Martellotto Luciano , Watkins David N. , Hannigan Gregory E.
Published: February 2011

The Hedgehog (Hh) signaling pathway is a highly conserved system for regulating metazoan development and cell fate. Integrin linked kinase (ILK) is required for proliferation of Hh-dependent neurons in the mammalian cerebellum, which also give rise to medulloblastoma, the most common childhood brain tumour. Sonic Hedgehog (SHh) binds to the Ptch transmembrane receptor, derepressing the Smo receptor to activate Hh signaling and key downstream targets such as Gli transcription factors. We find that SHh-induced osteogenic differentiation of C3H10T1/2 cells is blocked (>90% inhibition) by a small molecule ILK inhibitor, or by ILK-specific RNAi. SHh induces alkaline phosphatase (AP) activity in control 10T1/2 cells, but not cells transfected with ILK siRNA (>75% knockdown). Moreover we find that the small molecule ILK inhibitor suppresses SHh-induced differentiation in a dose-dependent manner, as effectively and to the same extent as the well-characterized selective Hh inhibitor, cyclopamine. Activation of Smo with the agonist, SAG, induces ILK-dependent AP induction in these cells, suggesting ILK acts downstream of Smo in the Hh pathway. SHh induction of the Gli1 transcription factor is also blocked by inhibiting ILK, suggesting ILK acts upstream of direct Hh targets. ILK inhibitor or siRNA suppresses constitutively activated Gli1 in medulloblastoma cells, derived from Ptch+/-:p53 compound heterozygote mice. In support of ILK involvment in Hh signaling, we have discovered a novel localization of ILK that indicates spatial proximity to Hh signaling molecules. The primary cilium is a microtubule-based organelle that is required for Hedgehog signaling, and we now find that ILK localizes in this structure. Using a selective ciliary marker, acetylated α-tubulin, we find that ILK localizes in the primary cilium along with components of the Hh pathway. Inhibition of ILK does not inhibit cilia formation, indicating that ILK is not involved in ciliogenesis. IFT proteins (eg. kinesin II motor subunit Kif3a), regulate both the assembly of, and the entry of Hh signaling proteins to, the cilium. Knockdown of Kif3a inhibits ciliogenesis and efficiently blocks SHh signaling, strongly suggesting the ciliary pool of ILK is critical for Hh signaling. Our work points to ILK as a novel therapeutic target in Hh-activated cancers.

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 995.
Abstract LB-112: The human papillomavirus type 16 E5 protein represses X-box binding protein 1 and cyclooxygenase-2 stress signaling in keratinocytes
Authors: Sudarshan Sawali R. , Liu Xuefeng , Schlegel Richard
Published: February 2011

Human papillomavirus (HPV) is the causative agent of 99% of cervical cancers, around 60% of which are caused by the high-risk type 16. While two of its three oncoproteins, E6 and E7, have been well characterized, the biological functions of the E5 protein remain elusive. Recent studies have suggested that E5 enhances HPV-induced oncogenesis, although in isolation it is not tumorigenic. In this study, we analyzed the gene expression changes induced by the endoplasmic reticulum (ER) resident HPV-16 E5 protein in primary human keratinocytes. Using real-time RT-PCR, we identified the downregulation of spliced X-box binding protein 1 (XBP-1), a key player in the ER-stress response, as a biochemical marker of E5 expression. The transmembrane kinase receptor responsible for the cleavage of XBP-1, ER-to-nucleus signaling 1 protein (IRE1α/ERN1), was also downregulated in E5-transduced cells. Furthermore, a microarray analysis of E5-transduced keratinocytes revealed the downregulation of another member of the ER-stress response pathway, prostaglandin G/H synthase and cyclooxygenase (COX-2/PTGS2). In addition, levels of COX-2 and XBP-1 were measured in keratinocytes expressing either low-risk HPV-6b E5 or a C-terminal 16E5 mutant containing a two amino acid mutation. The latter mutant had the histidine and alanine residues (conserved in the high-risk HPVs) replaced with tyrosine and isoleucine (conserved in the low-risk HPVs). Importantly, neither 6bE5 nor the H77YA78I 16E5 mutant was able to affect levels of these ER-stress response genes, suggesting that the ability to induce such changes in gene expression is specific to high-risk 16E5. Since preliminary data showed that expression of the E6 and E7 proteins in keratinocytes greatly induced XBP-1 splicing, we hypothesized that 16E5 might play a protective role against the host response to viral infection. In agreement with this postulate, we found that cells co-expressing E6, E7 and E5 had lower levels of COX-2 expression than cells expressing E6 and E7 alone. Interestingly, the levels of COX-2 in E6/E7-transduced cells were also supressed when compared to vector alone, suggesting that multiple pathways of COX-2 repression may exist. The mechanism by which E5 downregulates COX-2 is currently unknown, but it can be overcome by treatment with thapsigargin or tunicamycin, which initiate ER stress via calcium fluxes and abnormal protein glycosylation respectively. It is therefore unlikely that E5 is specifically tempering these pathways. Our data suggest a potential role for E5 in repressing the cellular ER-stress response following HPV infection.

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 LB-112.
Abstract LB-118: 17AAG, through hydrophobic binding, targets mitochondrial VDAC and inhibits cell invasion
Authors: Xie Qian , Wondergem Robert , Shen Yuehai , Cavey Greg , Ke Jiyuan , Thompson Ryan , Bradley Robert , Xu Yong , Holtrop Jennifer , Omar Hanan , Wenkert David , Xu Eric , Vande Woude George F.
Published: February 2011

17-allylamino-17-demethoxygeldanamycin (17AAG) is a member of the geldanamycin (GA) benzoquinone ansamycin family of anticancer drugs that is in clinical trials against several types of cancer. GA compounds bind to the ATP pocket of HSP90, and block its chaperone function resulting in the degradation of a wide variety of substrate oncoproteins, thereby providing a powerful “multi-target therapy”. GA compounds, however, are highly hydrophobic, requiring complicated drug vehicle formulation. In attempting to understand the influence of the hydrophobicity of GA drugs in vitro, we discovered that the hydrophobic molecules, GA or 17AAG, can bind directly to the highly hydrophobic voltage-dependent anion channel (VDAC) or to purified mitochondria where VDAC resides. Moreover, 17AAG binds to purified mitochondria prepared from all tissues and cells tested and compete with HSP90. Consistent with benzoquinone-ubiquinones functioning as classical Ca2+ mitochondrial regulators we associated the benzoquinone ring which all members of the GA family of drugs have as the responsible GA binding moiety with mitochondria. We show that both the ubiquinone, UbO, and GA drugs increase intracellular Ca2+, most likely through intracellular mitochondrial stores and both drugs diminish the plasma cationic current. By contrast, radicicol, with only an ansamycin ring, binds tightly to HSP90, but lacking the benzoquinone ring, has no measurable effect on cationic current or intercellular Ca2+ concentration. Thus, we propose that benzoquinone mitochondria binding through hydrophobic VDAC represents an alternative mode of action of GA drugs that can either alter HSP90 activity or be responsible for toxicity. Both activities must be considered when novel GA derivatives are developed as therapeutics.

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 LB-118.
Abstract LB-119: Production of functionally active glycoproteins and phosphoproteins based on a novel human in vitro expression system
Authors: Kilmer Greg , Vattem Krishna , Hommema Eric , Maas Ken , Deshpande Atul , Webb Brian L.
Published: February 2011

One of the key features of the human proteome is the abundance of post-translation modification (PTM), with more than 50% of proteins being glycosylated and more than 30% of all the proteins regulated by kinase activities. The alteration of protein glycosylation and other PTMs such as phosphorylation in cancer cells is closely related to the onset and progression of cancer and other disease states. In collaboration with RIKEN, we have developed a novel human in vitro translation (also known as cell-free expression) system, that can produce biologically active glycoproteins such as human chorionogonadotrophic hormone as well as active protein kinases such as Akt, MAPK and Aurora A kinase. A highly efficient PCR based approach will also be discussed that eliminates the need for cloning of genes into the expression vector and provides an easier expression of genes present in any other vector. In addition we demonstrate that our in vitro human expression, unlike the only other commercially existing mammalian expression system based on rabbit reticulocytes, can sustain expression of proteins for over 6 hours without the need to remove of inhibitory by products and is compatible with fluorimetric and colorimetric assays.

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 LB-119.
Abstract LB-133: Genome-wide differences in DNA methylation between tumor and normal adjacent tissue mapped by high-throughput sequencing of methyl-CPG binding domain based affinity-purified genomic fragments
Authors: Meredith Gavin D. , Dudas Miroslav , Krissinger Daniel , Marnellos George , Adams Christopher
Published: February 2011

Aberrant DNA methylation is characteristic of many cancers and differences in methylation have been observed in a wide variety of genomic contexts; for example, both within “classic” promoter-associated CpG islands and also in distal, non-CpG island regions. Establishing a method to broadly and efficiently survey the methylation patterns genome-wide in matched tumor and normal genomic DNA and to elucidate differences in these “methylomes” is the objective of the work presented. The method combines the power of methyl-CpG binding domain (MBD) proteins to sensitively and selectively bind methylated DNA sequences with the coverage, precision, and accuracy provided by high-throughput sequencing. Notably, MBD-affinity capture can also be used to sub-fractionate genomic DNA based on its average methyl-CpG content. In parallel, patient-derived matched samples of fragmented genomic DNA from a breast tumor and normal adjacent tissue were enriched and fractionated based on their methyl-CpG content with a commercial MBD-based affinity reagent (see figure) and high-throughput sequencing libraries were prepared from each fraction. The libraries were sequenced at 50 bp read-length on a SOLiD 3 Plus system and mapped to the human reference genome. Peak analysis of the distribution of mapped reads permitted the discovery of hundreds of putative differentially methylation regions (DMRs). Differential methylation at a number of these positions was confirmed by bisulfite-sequencing. We conclude that such enrichment and fractionation, when coupled to high-throughput sequencing, can be used to efficiently survey the majority of DNA methylation marks within samples of genomic DNA and to discover genomic loci of differential methylation. We conclude that such enrichment and fractionation, when coupled to high-throughput sequencing, can be used to efficiently survey a large fraction of all DMRs between samples of genomic DNA and to discover genomic loci of differential methylation. These datasets can then be compared with ChIP-seq and/or RNA-seq data sets to begin to decipher the functional genomics and epigenomics of cancer.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 LB-133.
Abstract LB-14: Endogenous expression of oncogenic PI3K mutation leads to accumulation of pro- and antiapoptotic proteins at mitochondria
Authors: Buron Nelly , Porceddu Mathieu , Torrance Chris , Disley Darrin , Borgne-Sanchez Annie
Published: February 2011

The phosphatidylinositol 3-kinase subunit PI3K is frequently mutated in human cancers and therefore represents an interesting therapeutic target. Cancer resistance to therapies is often related to expression of anti-apoptotic Bcl-2 family members which negatively regulate mitochondrial cell death. We investigated the influence of PI3K mutations on Bcl-2 family proteins both at the cellular and mitochondrial level. We used human breast epithelial cell lines with an endogenous “knock-in” PI3K mutation (HME-1 cells harbouring the H1047R activating mutation) from which we purified the mitochondria to homogeneity (95%). We first characterized these mitochondria for their sensitivity to reference compounds (Calcium, t-Bid) on 3 parameters: swelling, m loss and cytochrome c release. Mitochondrial preparations from wild-type and mutated HME-1 were analysed for their protein pattern in Bcl-2 family members. We observed accumulation of anti-apoptotic (Bcl-xl and Bcl-2) and pro-apoptotic (Bad) proteins in mitochondria isolated from PI3K mutated cells. We showed that the excess of anti-apoptotic proteins at the mitochondria does not change their sensitivity to the Bcl-2 family inhibitor, ABT-737. Investigations with PI3K-inhibitors are currently undergoing to determine the mitochondrial on-target biomarker for drug development.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 LB-14.
Abstract LB-140: An innovative approach for profiling of histone modification marks and gene expression across a panel of oncogenes and tumor suppressor genes in multiple cancer cell lines
Authors: Huang Jiaqiang , Quellhorst George J. , Gu Xiujing , Xiang Yan , Zeng Xiao , Boon Kathy
Published: February 2011

Objectives: Histone modification marks and gene expression profiling on multiple human oncogenes and tumor suppressor genes to study the correlation between epigenetic modifications and gene expression in cancer cells derived from different tissue/cell types.

Methodology: Chromatin Immunoprecipitation (ChIP) was performed on the isolated chromatin samples from various cancer cell lines (MCF7, A549, HepG2, HCT116, PC3 and HeLa) using antibodies against H3K4me2, H3K4me3, H3Ac, H3K27me3, H3K9me3, H4K20me3, and RNA Pol II. Each resulting ChIP DNA fraction was analyzed with a ChIP Real-Time PCR Array consisting of specific PCR primers targeting the downstream region of the transcription start site (within + 1Kb). For this study 84 oncogenes & tumor suppressor genes were measured simultaneously along with control gene regions characteristic for each histone modification. Real-time RT-PCR Arrays were also employed to analyze the mRNA levels for all 84 genes in the same 6 cancer cell lines. These results allow analysis to correlate specific histone marks with the transcript levels.

Results: The data indicate a much more complex correlation between modified histone marks and gene expression across different cancer cell lines. The results provide insights into the complex epigenetic regulation in cancer cells. The combination of ChIP-qPCR Arrays (histone modifications) and RT-qPCR Arrays to study multiple oncogenes and tumor suppressor genes simultaneously in various cancer samples should prove a valuable and fast approach in the assessment of the correlation between epigenetic regulation and clinical characteristics.

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 LB-140.
Abstract LB-151: Targeted gene resequencing in a K-ras-dependent mouse model of metastatic lung adenocarcinoma
Authors: McFadden David G. , Xuan Zhenyu , Rosebrock Adam , Politi Katerina , Santiago Philip , Hodges Emily , Winslow Monte , Hiramatsu Nobuhito , Pirun Mono , Crowley Denise , Bronson Roderick , Socci Nicholas , Varmus Harold , Hannon Greg , Jacks Tyler
Published: February 2011

Progression of lung adenocarcinoma to invasive, metastatic disease is responsible for the majority of deaths from lung cancer and remains incompletely understood. We have developed a genetically-engineered mouse model of lung adenocarcinoma initiated by mutant K-ras and loss of p53 that develops invasive, metastatic disease. In order to identify the genes and pathways that cooperate with oncogenic K-ras and loss of p53 to contribute to tumor development, progression, and metastasis, we have utilized in situ exon capture and massively parallel DNA sequencing to identify somatically-acquired point mutations in advanced stage murine lung adenocarcinoma cell lines. We have initially chosen a target set of 1283 cancer-relevant genes to develop our capture, sequencing, and bioinformatics platforms. Using tumor cell lines derived from K-ras-dependent tumors, we have successfully developed and refined these methods and have identified somatic mutations in our test samples. These mutations have been validated by conventional methods in the tumor cell line and founder tumor DNA. We have developed a production phase target gene set consisting of 1909 cancer-relevant genes and have initiated capture and sequencing of additional murine lung adenocarcinoma cell lines and tumors. We believe these datasets will expand the understanding of the biology of tumor progression and serve as an important biological filter for human cancer gene re-sequencing efforts.

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 LB-151.
Abstract LB-157: A murine model of Fanconi anemia-associated head and neck cancer
Authors: Hays Laura E. , Bagby Grover C.
Published: February 2011

Fanconi anemia (FA) predisposes patients to aggressive head-and-neck-squamous-cell-carcinomas (HNSCCs) early in life. These tumors are particularly difficult to treat because of the underlying hypersensitivity of all somatic cells from these patients to cross-linking agents. An in vivo model is thus badly needed to delineate the unique molecular mechanisms involved in tumor formation and to identify opportunities to exploit this mechanism therapeutically. Although mice with inactivating mutations of FA genes have been developed, these mice do not spontaneously develop HNSCCs. Therefore, we sought to develop our own model by treating wild-type (WT) and Fancc-deficient (FA-C) mice with the carcinogen 4-nitroquinoline 1-oxide for 45 weeks. Although tumor incidence, histological type and grade, and anatomical location were similar in WT and FA-C mice, cells isolated from FA-C tumors displayed higher levels of endogenous chromosomal instability compared to cells from WT tumors. None of the mice developed metastases, most likely because they required early euthanasia as tumor formation inhibited the ability to eat and drink. To compare the metastatic potential of WT and FA-C HNSCC cells, we subcutaneously injected these cells lines into the flanks of immunocompetent (C57Bl/6J) and immunodeficient (NOD/SCID) mice. Interestingly, tumors only formed in NOD/SCID mice indicating immune-mediated suppression of tumor growth in the immunocompetent mice. In NOD/SCID mice, 4 out of 6 WT and 3 out of 4 FA-C cell lines induced tumor formation. However, all of the tumor-forming FA-C lines produced aggressive tumors that penetrated into the retro-peritoneal space and/or produced hind-leg paralysis, while only 1 out of 4 WT lines invaded to this extent (a phenomenon that occurred in fewer than 25% of mice injected with this line). In addition, we found that tumor forming ability of FA-C HNSCC cell lines did not require loss of Fancc because FA-C lines transduced with wild-type Fancc remained fully tumorigenic in vivo. In conclusion, we have developed a model of FA related HNSCCs utilizing FA and WT cell lines. The model permits the outgrowth of tumors that are histologically, genomically, and biologically similar to corresponding tumors in humans. Currently we are utilizing this model to test the efficacy of small molecules in inhibiting FA tumor progression and are testing the role of the tumor microenvironment and the immune system in modulating the development of FA-related HNSCC.

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 LB-157.
Abstract LB-17: WW domain-mediated interaction with Wbp2 is important for the oncogenic property of TAZ
Authors: Chan Siew Wee , Lim Chun Jye , Huang Caixia , Chong Yaan Fun , Harvey Kieran , Hong Wanjin
Published: February 2011

The transcriptional co-activators YAP and TAZ are downstream targets inhibited by the Hippo tumor suppressor pathway. YAP and TAZ both possess WW domains which are important protein-protein interaction modules that mediate interaction with proline-rich motifs, most commonly PPXY. The WW domains of YAP play complex regulatory roles as exemplified by two recent reports showing that they can positively or negatively influence YAP activity in a cell and context-specific fashion. In this study, we show that the WW domain of TAZ is important for it to transform both MCF10A and NIH3T3 cells and to activate transcription of ITGB2 but not CTGF, as introducing point mutations into the WW domain of TAZ (WWm) abolished its transforming and transcription-promoting ability. Using a proteomic approach we discovered potential regulatory proteins that interact with TAZ WW domain and identified Wbp2. The interaction of Wbp2 with TAZ is dependent on the WW domain of TAZ and the PPXY-containing C-terminal region of Wbp2. Knockdown of endogenous Wbp2 suppresses, whereas overexpression of Wbp2 enhances, TAZ-driven transformation. Forced interaction of WWm with Wbp2 by direct C-terminal fusion of full-length Wbp2 or its TAZ-interacting C-terminal domain restored the transforming and transcription-promoting ability of TAZ. These results suggest that the WW domain-mediated interaction with Wbp2 promotes the transforming ability of TAZ.

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 LB-17.
Abstract LB-173: Statin use and colorectal adenoma risk: Results from the Adenoma Prevention with Celecoxib (APC) trial
Authors: Bertagnolli Monica Marie , Hsu Meier , Hawk Ernest T. , Eagle Craig J. , Zauber Ann G.
Published: February 2011

Discussant: John A. Baron, Dartmouth Medical School, Lebanon, NH.

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 LB-173.
Abstract LB-189: BRAFdeletion and pharmacological inhibition enhance K-ras driven tumorigenesis
Authors: Murriel Christopher L. , Chen Honglin , Kenski Denise , Bou-Reslan Hani , Yuan Wenlin , Liang Yuxin , Carano Richard , Couto Suzana , Seshagiri Somasekar , Davis David P. , Johson Leisa
Published: February 2011

Discussion

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 LB-189.
Abstract LB-191: Loss of the wild-type Kras allele enhances the cellular proliferation and metastasis of pancreatic ductal adenocarcinomas in a conditional P16 and Kras mutant mouse model
Authors: Qiu Wanglong , Sahin Fikret , Iacobuzio-Donahue Christine A. , Garcia-Carracedo Dario , Wang Wendy M. , Arking Dan E. , Lowy Andrew M. , Hruban Ralph H. , Remotti Helen E. , Su Gloria H.
Published: February 2011

Discussion

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 LB-191.
Abstract LB-192: In vivo zebrafish tyrosine kinase assay for anti-cancer drug screening
Authors: Li Chun-Qi
Published: February 2011

Discussion

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 LB-192.
Abstract LB-197: Tumor vasculature associated marker as a target for recombinant DNA based vaccines
Authors: Facciabene Andrea , Facciponte John , Coukos George
Published: February 2011

Discussion

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 LB-197.
Abstract LB-202: STAT3 induces pituitary tumor transforming gene (PTTG) expression
Authors: Zhou Cuiqi , Ren Song-Guang , Tong Yunguang , Wawrowsky Kolja , Bannykh Serguei , Melmed Shlomo
Published: February 2011

PTTG is overexpressed in multiple human tumors, but mechanisms regulating tumor PTTG abundance remain elusive. As EGF induces pituitary cell PTTG expression, and as STAT3 is required for pituitary cytokine responses, we investigated STAT3 regulation of PTTG. STAT3 and PTTG were both concordantly overexpressed in 79 human pituitary tumor specimens (62% and 56% respectively; p<0.01) as assessed by confocal immunofluorescence. We therefore treated ACTH-secreting AtT20 corticotroph tumor cells with IL-6 which dose-dependently increased STAT3 phosphorylation, PTTG and POMC expression. STAT3 also specifically bound to the human PTTG promoter (−2000 to −3000 bp) and induced PTTG transcriptional activity (2.2-fold) as assessed by chromatin immunoprecipitation and luciferase reporter assay, implying that PTTG acts as an intracellular STAT3 target. To elucidate the function of this pathway, we constructed WT STAT3, constitutively activated STAT3 (STAT3-C) and dominant negative STAT3 (STAT3-DN) plasmids, and selected respective stable transfectants. STAT3 transfection doubled PTTG mRNA and protein abundance, while PTTG induction was enhanced 3-fold in STAT3-C, and totally abrogated in STAT3-DN transfectants (0.7-fold). Stable STAT3 overexpression also enabled in vitro colony formation which was strongly attenuated by PTTG RNAi (∼50%, p<0.01). Elevated STAT3 expression, or STAT3 activation, both increased HCT116 cell migration and invasion 5-fold as measured by in vitro cell assays, whereas cell mobility was abolished by STAT3-DN (>90%). Knockdown of PTTG expression by RNAi strongly suppressed STAT3-induced cell migration and invasion by up to 80%. Attenuating PTTG expression by RNAi in STAT3-C stable cells constrained downstream myc, cyclin D3, K-Ras and MMP expression. Moreover, STAT3 inhibitor VI dose-dependently (10-150 M) suppressed STAT3 phosphorylation and subsequently inhibited PTTG, cyclin D3, POMC expression and ACTH secretion as treated for 24 hours. We also generated PTTG RNAi stable transfectants on STAT3-C stable cells and injected these compound transfectants s. c. to nude mice. PTTG RNAi decreased STAT3-C-induced in vivo tumor volume (32%, p<0.01), compared to injected scrambled control transfectants which readily formed tumors. These results elucidate a mechanism for tumor PTTG abundance, whereby STAT3 activation induces PTTG expression to facilitate tumor formation. These findings further elucidate PTTG actions in tumor progression and support the rationale for targeting PTTG to abrogate endocrine tumor growth.

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 LB-202.
Abstract LB-204: Gene expression profiling of hormone-regulating pathways in tumoral and peritumoral breast tissues
Authors: Christine Flageole , Audet-Walsh Étienne , Bélanger Alain , Guillemette Chantal
Published: February 2011

Purpose: The purpose of our investigation was to study the mRNA expression of sex-steroids pathways involved in androgen and estrogen synthesis and metabolism in tumor and surrounding peritumoral normal breast tissues. High integrity RNA was purified and specific quantitative realtime PCR assays was performed to assess expression level of thirty two genes in breast tissues from the 4 quadrants of postmenopausal mastectomy patients diagnose with breast cancer. The expression changes between tumors (n=31) and paired normal (n=86) tissues were assessed using a general linear model on log-transformed data.

Results: The main pathway involved in estrogen synthesis in postmenopausal women, steroid sulfatase, was detected at lower levels in tumor tissues compared to non-malignant tissues. mRNA expression levels of aromatase converting androgen to estrogen were relatively constant between peritumoral normal and tumoral tissues while androgen conjugating enzymes leading to inactive metabolites were overexpressed in tumoral tissues (1.4-fold; P < 0.04). Expression of CYP1A1, involved in the synthesis of 2-hydroxy-catecholestrogens, was constantly detected at lower level in tumors compared to peritumoral tissues (3.7 fold; P < 0.001). However, no prominent changes for the catechol-o-methytransferase (COMT), involved in the conversion of 2-hydroxy-catecholestrogens to methoxy metabolites with cancer-protective properties, was noted. In contrast, CYP1B1 expression was enhanced by 1.7 fold in tumors (P < 0.001) suggesting a greater in situ formation of 4-hydroxy-catecholestrogens, a group of metabolites associated with DNA damage and production of redox species. Data also reveal a lower expression in tumors of the NAD(P)H quinone oxydoreductase 2 (NQO2) most active against catechol estrogens (1.8-fold; P=0.04), while expression of NQO1 remained unchanged. These observations were further enhanced in estrogen receptor positive tumors.

Conclusion: Our data add to the emerging evidence of an imbalance in the production of androgen, estrogen and their metabolites in breast tumors, preferentially leading to the accumulation of carcinogenic metabolites while decreasing protective metabolites in neoplastic tissue compared to normal tissue.

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 LB-204.
Abstract LB-217: A universal cancer detection platform which measures telomerase activity from live circulating tumor cells captured on microfilter
Authors: Xu Tong , Lu Bo , Tai Yu-Chong , Goldkorn Amir
Published: February 2011

Objective. · Circulating tumor cells (CTCs) quantified in cancer patients’ blood can predict disease outcome and response to therapy. However, the CTC analysis platforms currently used are expensive, labor-intense, operator-dependent, and only applicable to tumors of epithelial origin. To address these limitations, we engineered a universal cancer detection platform which measures telomerase activity from live circulating tumor cells captured on microfilter.

Methodology: Microfilters were fabricated from parylene-C by a multi-step Reactive Ion Etching (RIE) technique and then connected to a constant low-pressure fluid delivery system. To optimize slot size and filtration pressure, PC3 prostate cancer cells were stained with live-cell dye (Calcein-AM) and spiked into whole blood. After filtration, the captured cells were counter-stained with dead-cell stain (propidium iodide, PI) and counted to determine capture efficiency, viability, and enrichment. Telomerase activity measurement was accomplished using blood spiked with cancer cell lines as well as actual patient samples. Briefly, 7.5 ml blood samples were depleted of red blood cells by ficoll gradient centrifugation, and the PBMC fraction was resuspended in PBS, captured on microfilter and lysed. Telomerase activity was assayed from the cell lysates using a modified quantitative PCR telomere repeat amplification protocol (qPCR-TRAP).

Results: The optimized microfilter platform was capable of cell capture from 7.5 ml of whole blood in under 15 minutes, achieving 80% capture efficiency, 90% cell viability and 1500-fold sample enrichment. Importantly, the captured cells retained normal morphology by scanning electron microscopy and could be readily manipulated, further analyzed, or expanded on- or off-filter. Remarkably, telomerase activity was reliably detected by qPCR-TRAP from as few as 25 cancer cells spiked into 7.5 ml whole blood, as well as from actual CTCs in patient blood samples and even from single cancer cells captured on microfilter.

Conclusions: We present a novel cancer biomarker platform capable of assaying telomerase activity in live CTCs captured on microfilter from human blood. This new technology can be used to elucidate the basic biology of cancer dissemination and also applied clinically to improve prognostication, treatment, or even early detection of common malignancies. It is fast and simple yet fully quantitative and applicable to nearly all solid tumor types, making microfilter telomerase analysis a truly universal and highly promising cancer biomarker platform.

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 LB-217.
Abstract LB-222: Identification of rare KRAS codons 12 and 13 mutations by shifted termination assay
Authors: Doong Howard , Li Xiaofei , Chen Caifu , Walker Julian
Published: February 2011

We have developed a sensitive and specific method for research studies, using Shifted Termination Assay (STA) to detect 12 possible KRAS mutations in codons 12 and 13. In this study, we analyzed 118 samples collected from formalin-fixed and paraffin-embedded (FFPE) metastatic colorectal cancer tumors.

The detection method includes carrying out a primer extension reaction in the presence of a wild-type DNA sequence. The primer extension reaction is stopped when encountered by a mutated base. If no mutation is detected the reaction terminates at the next nucleotide resulting in a large wild-type fragment. This creates a shift in the amount of labeled nucleotides incorporated on the primer extended product, and is then differentiated by fragment analysis using Applied Biosystems capillary electrophoresis systems.

Among the 118 samples tested, 32 were found to carry a mutation with 9 different variants. The most common mutations found in codon 12 were GGT>TGT (G12C, 9/32) and GGT>GTT (G12V, 8/32). Two rare variants in codon 12, GGT>GCT (G12A, 1/32) and GGT>CGT (G12A, 1/32), were also observed. For codon 13, the most common mutation found in the samples was GGC >GAC (G13D). In this study, we found 4 variants with 3 different types - 2 samples of GGC>AGC (G13S), 1 sample of GGC>GAC (G13D) and 1 sample of GGC>CGC (G13R). In summary, our data suggest that more KRAS mutations were observed in codon 13 relative to codon 12 in research samples, and the STA assay is an easy to use, robust method to detect possible KRAS mutations in codons 12 & 13. STA is currently for research use only, not for use in diagnostic procedures.

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 LB-222.
Abstract LB-228: YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway
Authors: Zhang Jianmin , Ji Jun-Yuan , Yi Min , Overholtzer Michael , Smolen Gromoslaw A. , Wang Rececca , Brugge Joan S. , Dyson Nicholas J. , Haber Daniel A.
Published: February 2011

The Hippo signaling pathway was initially identified in Drosophila and components in this pathway are highly conserved in mammals. We and others have recently shown that the Hippo pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP, a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation. To date, studies of YAP target genes have focused on cell-autonomous mediators. Here, we show that YAP-expressing MCF10A breast epithelial cells secrete growth factors that enhance the proliferation of neighboring untransfected cells, implicating a non-cell autonomous mechanism. Using cytokine and growth factor array analysis, we identified the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG), as a transcriptional target of YAP, whose induction contributes to YAP-mediated cell proliferation and migration. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1/2 is sufficient to induce AREG expression, consistent with a physiological regulation of AREG by the Hippo pathway. Furthermore, genetic interactions between the Drosophila YAP orthologue Yki and Egfr signaling components support the link between these two highly conserved signaling pathways. In conclusion, YAP-dependent secretion of AREG implicates activation of EGFR signaling as an important non-cell autonomous effector of the Hippo pathway, with relevance for the regulation of both physiological and malignant cell proliferation.

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 LB-228.
Abstract LB-239: The Rho GEF, GEF-H1, is regulated by and activates Rho in response to 3-D collagen matrix density in breast epithelial cells
Authors: Jessica Heck N. , Ponik Suzanne M. , Provenzano Paolo P. , Keely Patricia J.
Published: February 2011

Cell interactions with the extracellular matrix (ECM) have a profound influence on carcinoma progression, invasion, and metastasis. We and others have established that breast epithelial cells sense and respond to the stiffness of their ECM environment using the small GTPase Rho, however the exact signaling mechanism has not been identified. In addition to Rho, focal adhesion kinase (FAK) has been linked to mechanosensing, and studies from our lab have revealed FAK as a key mediator of breast tumor progression. We have found that mammary specific deletion of FAK results in tumors that are neither locally invasive nor metastatic. Expression analysis from FAK−/− tumors demonstrated that a number of Rho regulatory molecules were regulated by FAK. Of particular interest, mRNA for the Rho GEF, GEF-H1, was significantly decreased in FAK−/− mammary tumors. While relatively little is known about GEF-H1, it has been implicated in the crosstalk between microtubules and the actin cytoskeleton, positioning it as a potential regulator of Rho-mediated contractility. While it is established that Rho activity is regulated by matrix stiffness, Rho regulatory molecules that modulate signaling in 3D matrices, and specifically in response to changes in matrix stiffness, have yet to be identified. Here we demonstrate that the Rho specific exchange factor, GEF-H1, is activated by and in turn regulates Rho activation as well as Rho-mediated contractility in breast epithelial cells in response to 3D matrix density. Because we have demonstrated that extracellular signal-regulated kinase (ERK) is activated in response to increased 3D collagen matrix density, and others have shown that ERK signaling modulates GEF-H1 exchange activity for RhoA, we are investigating whether ERK mediates mechanosignaling to GEF-H1. Additional studies are underway to determine the role of GEF-H1 in 3D breast cell invasion.

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 LB-239.
Abstract LB-241: Cancer-associated fibroblasts modulate growth- and metastasis-promoting pathways in human pancreatic cancer cells and augment orthotopic tumor growth in mice
Authors: Walters Dustin M. , Adair Sara J. , Stokes Jayme B. , Lowrey Bryce , Parsons J. Thomas , Bauer Todd W.
Published: February 2011

Background: The role of cancer-associated fibroblasts (CAFs) in the development, progression, and metastasis of pancreatic ductal adenocarcinoma (PDAC) is not completely understood. We sought to better characterize the role of CAFs by evaluating the effect of co-culturing CAFs and pancreatic cancer cells on growth and metastasis pathways.

Methods: Resected human PDACs were grown orthotopically in mice. From these tumors, human pancreatic cancer cells and mouse CAFs were isolated and grown in culture. Three cell culture environments were created: 1) pure (>99%) human pancreatic cancer cells, 2) pure (97%) mouse CAFs, and 3) co-culture of both cell types. IHC and FACS were used to confirm cell type and species of origin. Phospho-receptor tyrosine kinase (pRTK) and cytokine arrays were performed on the three groups. Additionally, the effect of CAFs on PDAC growth was evaluated in nude mice by orthotopically injecting cancer cells (1 × 106 cells) with and without CAFs (1 × 106 cells).

Results: CAFs stained positively for -smooth muscle actin (-SMA) but were negative for E-cadherin, while cancer cells stained positively for E-cadherin, but not -SMA. FACS for MHC-I confirmed mouse origin of the CAFs (negative) and human origin of the cancer cells (positive). Co-culture of mouse fibroblasts and human tumor cells led to significant upregulation of cell proliferation pathways (evidenced by increased human pRTKs and cytokines, including EGFR1, EGFR2, c-Met, Ron, IGF-1R, and thrombospondin-2), angiogenic pathways (evidenced by increased secretion of VEGF, IL-8, and angiogenin) and matrix degradation and remodeling pathways (increased secretion of MMPs 8 and 9, TIMPs 1 and 4, and uPA). Orthotopic mouse injection of human pancreatic cancer cells in combination with mouse CAFs (1:1 ratio) led to larger tumors at 3 weeks as compared to injection with cancer cells alone (1129 mm3 vs. 443 mm3, p < 0.005).

Conclusions: Using co-culture of CAFs and pancreatic cancer cells we have recapitulated the modulation of pathways responsible for cell proliferation, angiogenesis, and matrix remodeling thought to occur in vivo during tumor progression. Further, we show that co-injection of CAFs and pancreatic cancer cells significantly augments orthotopic tumor growth in mice. These studies lay the groundwork for future studies to define the critical factors produced by stromal fibroblasts and the role of such factors in PDAC progression, allowing new insights into improving patient therapy.

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 LB-241.