bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4192: LIN28 mRNA targets in breast cancer cells
Authors: Kinyamu Harriet K. , Yang Jun , Archer Trevor K.
Published: August 2012

LIN28 is a RNA binding protein and a pluripotency factor. It represses tumor suppressing microRNA let-7 and activates let-7 oncogene targets such as HMGA2 and c-Myc. Recent reports show that LIN28 overexpression promotes tumor cell migration and cellular transformation through repression of let-7, and that overexpression is associated with advanced stages of disease, including breast cancer. The molecular function and mode of action of LIN28 in breast cancer is not yet elucidated. To further understand the function of LIN28, we explored LIN28 mRNA targets in breast cancer cells using RNA-protein immunoprecipitation (RIP) followed by RNA sequencing (RIP-Seq). Only 418 genes (1.7% of the human total genes) are enriched more than 1.0 RPKM (reads per kilobase of exon model per million mapped reads) in the LIN28 RIP relative to control RIP. LIN28 mRNA targets in breast cancer cells function in processes that drive tumorigenesis, including cell proliferation, inflammation and metastasis. Therapeutic drugs inhibiting LIN28 could be promising in treatment of cancer. Our experiments also demonstrate that MG132, a proteasome inhibitor, inhibits LIN28 expression and modulates expression of LIN28 mRNA targets in breast cancer cells. Studies are ongoing to reveal the mechanisms by which LIN28 regulates bound targets and how proteasome inhibitors impact this regulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4192. doi:1538-7445.AM2012-4192
Abstract 4195: Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E-BP1
Authors: Liu Jianyu , Gao Tianyan
Published: August 2012

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Deregulation of translation initiation leads to tumor development and progression. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Hyperphosphorylation of 4E-BP1, which leads to inactivation of 4E-BP1, has been linked to human cancers with increased malignancy. While it has been demonstrated previously that the phosphosphorylation of 4E-BP1 is controlled by mTOR in the mTOR complex 1 (mTORC1), the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1 through an shRNA library screen of Ser/Thr protein phosphatases. Co-immunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells, and purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 and SW480 cells results in an increase in the phosphorylation of 4E-BP1 at both Thr37/46 and Ser65 sites. This PPM1G-mediated dephosphorylation of 4E-BP1 is specific as the phosphorylation of upstream regulators of 4E-BP1 including Akt and mTOR is not altered in PPM1G knockdown cells. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mTOR inhibitor treatment is significantly slowed down in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when expression of PPM1G is depleted. As the result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of protein translation by negatively controlling the phosphorylation of 4E-BP1. Thus, PPM1G may serve as a tumor suppressor by directly counteracting mTOR to inhibit cell growth in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4195. doi:1538-7445.AM2012-4195
Abstract 4196: Identification and investigation of ethnic specific gene expression differences in non-cancerous breast tissue
Authors: Baumbach-Reardon Lisa L. , Gomez Carmen , Ahearn Mary Ellen , Green Ashley , Ellison Kevin , Yariz Kemal O. , Issac Biju , Clarke Jennifer , Ambs Stefan , Pegram Mark
Published: August 2012

Disparities in breast cancer (BC) stage of presentation and survival are well documented in patients of different ethnicities. These differences are undoubtedly due to a combination of extrinsic and intrinsic factors. Our group is focused on the genetic contributions to these disparities. Recent observations by our group and others suggest not only the involvement of specific genes contributing to BC risk in African-American (AA) women, but that multiple distinct ethnic-specific gene expression patterns exist in both matched cancerous and normal breast tissue samples. We report the first results of a parallel study which is addressing an unanswered question: are there ethnic-specific differences in the transcriptome of normal breast tissue? We are completing gene expression profiling studies in normal breast FFPE samples from 25 AA and 25 Caucasian (Cauc) women with no history of breast cancer. Laser capture micro-dissection (LCM) is used to further identify and extract epithelial vs stromal cells from each FFPE slide for use in RNA expression profiling. RNA isolation and hybridization occurs in collaboration with Almac Diagnostics (Dx) using their proprietary Breast Cancer DSA array. Gene expression data analyses involves multiple statistical packages developed for DNA microarray studies, and is focusing on comparison of gene expression profiles across four study groups- AA vs Cauc samples and epithelial vs stroma cells. For baseline comparison, a subset of this study group (10 AA and 10 Cauc samples non-LCM dissected) has also been analyzed. Results from these non-LCM dissected samples displayed expression differences between AA and Cauc in 333 known genes. Applying the stringency criteria of a fold change of two or greater and a statistical p-value of 0.01, reduced the significant gene list to 61 genes. Initial pathway analyses indicate these genes are involved in cell adhesion, G-protein signaling, apoptosis and survival pathways, and cytoskeleton remodeling - all critical pathways for cell survival. These initial results are being validated in the LCM-dissected samples, and our latest results will be presented. Results from analyses of these normal samples will be compared to our previous results obtained in matched tumor-normal triple negative AA and Cauc breast cancer samples (as well as publicly available data sets). Combined analysis of tumor and normal breast expression (transcriptome) data will help to better understand the possible significance of gene expression differences in breast tissue between ethnic groups.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4196. doi:1538-7445.AM2012-4196
Abstract 4197: Discovering FUS-CHOP targets: A Chip-Seq approach
Authors: Tonin Elena , Sirocco Francesco G. , Rossi Sabrina , Tos Angelo Paolo Dei , Libra Massimo , Hannon Gregory J. , Maestro Roberta
Published: August 2012

Myxoid liposarcoma (M-LS) is one of the most common soft tissue sarcomas in adults. Approximately 90% of cases show the characteristic t(12;16)(q13;p11) translocation, which produces the FUS-CHOP (FC) oncogene. FUS is a RNA-binding protein expressed in several tissues; CHOP a bZIP transcription factor involved in adipocyte differentiation. Although it is known that CHOP bZIP region is essential for FC-induced transformation, little is known about FC transcriptional targets and how FC participates in the development and progression of M-LS. Moreover, it is unclear whether FC controls the transcription of a set of genes different from the set controlled by CHOP. Understanding how the FUS-CHOP fusion protein causes M-LS will aid in the development of novel targeted therapies. To shed light on this issue we sought to exploit the ChIP-Seq technique (Chromatin Immunoprecipitation followed by next generation sequencing). To this end, chromatin immunorecipitation was performed on cell lines expressing FC (endogenous or ectopic). DNA co-precipitated together with the FC protein was analyzed by using an Illumina GAIIX platform. FC-bound DNA was mapped to the genome using the aligner Bowtie and the enriched regions were identified by using the MACS peak caller. ChIP-Seq data were combined with RNA-seq analysis to screen for the potential targets. Our approach was validated by the identification of LONP1, CLCN3 and GADD34, which have been previously reported to be modulated by CHOP. The most interesting candidates will be further investigated for their implication in FC development and progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4197. doi:1538-7445.AM2012-4197
Abstract 4199: CDKN2A/p16INK4a 5′UTR variants in melanoma predisposition: Lost in translation, somewhere
Authors: Bisio Alessandra , Andreotti Virginia , Latorre Elisa , Vescovo Valerio Del , Ciribilli Yari , Provenzani Alessandro , Quattrone Alessandro , Bianchi-Scarra' Giovanna , Denti Michela A. , Ghiorzo Paola , Inga Alberto
Published: August 2012

The CDKN2A gene is the most common high penetrance susceptibility gene identified to date in melanoma families. While functional tests for determining the pathogenicity of missense germline mutations in the CDKN2A coding region have been developed, rare polymorphisms or sequence variants at the CDKN2A/ p16INK4a 5′UTR, encountered during routine screening, are usually defined as variants with unknown significance after determining their frequency in control population and the cosegregation analysis in the family, when possible. We recently developed reporter assays to study a panel of p16INK4a 5′UTR variants identified as heterozygous changes in patients from a hospital-based series of melanoma cases (c.-21C>T; c.-25C>T&c.-180G>A; c.-56G>T; c.-67G>C). Monocistronic as well as bicistronic luciferase-based reporter vectors were developed and used to test wild type and variant p16INK4a 5′UTR activity upon transient transfection in melanoma-derived cells (WM266-4, G361 and SK-Mel-5) and in the breast cancer-derived MCF7 cells. Results revealed that the c.-21C>T variant had a strong negative impact on the reporter activity, similar to that of the known melanoma-predisposing mutation c.-34G>T, included as a control. The variants at –56 and at –25&-180 exhibited a milder impact, while results with c.-67G>C were dependent on the type of reporter vector. Quantification of the luciferase mRNA conducted in parallel with the luciferase assay indicated that the impact of the variants was mainly post-transcriptional. We also applied a polysomal profiling technique to measure allelic imbalance starting from heterozygous patient-derived cell lines and found that the c.-21C>T variant but also c.-56T>G and c.-67G>C exhibited lower association with the polysomes suggestive of reduced mRNA translation efficiency. A panel of eleven additional germline variants in the 5′UTR of p16INK4a is being investigated. In particular we are focusing on the functional interactions between wild type and variant p16INK4a 5′- and 3′-UTR sequences and on the impact the variants can have on the targeting of the p16INK4a mRNA by microRNAs (including mir-346, -636, -639, -935) or RNA binding proteins (including YB-1).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4199. doi:1538-7445.AM2012-4199
Abstract 42: Expression of interleukin-7 and its signalling intermediates in human neuroblastoma tumours.
Authors: Prasad Linda , Gayagay Andrew , Charlton Amanda , Henwood Anthony , Graf Nicole , Arbuckle Susan , Catchpoole Daniel R.
Published: August 2012

Neuroblastoma (NB) is the most common extracranial childhood malignant solid tumour which shows remarkable biological heterogeneity. It has been speculated that schwann cells influence NB tumour growth, by secreting as yet to be identified ‘soluble factors’ that serve as anti-proliferative and differentiation signals for neuronal cells. Several gene expression studies indicate that interleukin 7 (IL7) has increased expression in NB specifically in tumours with a better prognosis, making IL7 a key candidate for this soluble factor. In this study, the expression of IL7, IL7 receptor (IL7R) and its downstream signalling proteins, including the Janus Kinases (JAK1 and JAK3), Signal Transducers and Activators of Transcription (STAT5) and Phosphorylated STAT5 (pSTAT5) were analysed using immunohistochemical stains in a cohort of 100 patients with stroma poor NB and 24 with stroma rich ganglioneuroma (GN). The expression of these proteins were correlated to the expression of the diagnostic markers S100 (stromal marker), NB84 (neuroblast marker) and CD99 (negative marker for NB). The immunohistochemical analysis showed that IL7 expression was strongly positive exclusively in schwannian stroma in both favourable and unfavourable histology whilst staining ganglionic cells as well. The levels of expression, as quantified by specialised digital image analysis algorithms (Aperio, USA), revealed IL7, IL7R, JAK1, STAT5 and pSTAT5 were all increased in GNs compared to NBs. There was no correlated change in expression levels of JAK3 in the tumours. Expression of pStat5 was found to be significantly reduced (t-test, p=0.00013) in stroma poor NB compared to GN. Our findings implicate IL7 within the schwannian stroma of the tumour architecture as having a paracrine signalling effect on neighbouring neuroblasts which may provide the anti-proliferative and differentiation signals postulated. Further investigation of the importance of IL7 in neuroblastoma will define the biological significance of the IL7-Stat5 signalling cascade and whether targeting the specific IL7 signalling pathway proteins will raise novel treatment strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 42. doi:1538-7445.AM2012-42
Abstract 420: Physiological β-catenin signaling regulates pluripotency genes in cancer microcell hybrids
Authors: Cheng Yue , Cheung Arthur Kwok Leung , Ko Josephine Mun Yee , Phoon Yee Peng , Lung Maria L.
Published: August 2012

Both β-catenin signaling and Nanog were previously reported to be involved in cell fusion-mediated somatic cell reprogramming. It remains unclear as to how the β-catenin signaling pathway is initiated and whether this pathway may directly control the expression of core stem cell factors such as Nanog and Oct4. Since β-catenin signaling is a predominating force for the regulation of cellular fate and basic levels of this signaling are needed for somatic cell reprogramming, we speculate that transfer of a single copy of chromosome 3, where β-catenin maps and is controlled by its natural regulators, into somatic cancer cells may appropriately induce this pathway and switch on the expression of endogenous pluripotency genes in recipient cells. We previously generated human nasopharyngeal (HONE1) and esophageal (SLMT1) carcinoma hybrid cells with an intact copy of transferred chromosome 3. At both transcriptional and translational levels, we detected obvious expression of genes related to the Wnt/β-catenin pathway in HONE1/chromosome 3 hybrid cells. In these hybrid cells, β-catenin, c-Myc, Axin2, Tcf1, Sox2, Klf4, Oct4, and Nanog were either strongly expressed or up-regulated. The loss of region that contains B-catenin locus in transferred chromosome 3 abolishes the expression of core stem cell genes. The control of these gene activities by physiological β-catenin signaling was further confirmed in HONE1/chromosome 17 hybrids that contain the downstream components of β-catenin signaling Axin2 and Stat3, but was not detected in hybrids with an irrelevant transferred chromosome and SLMT1 cells that had a relatively high level of endogenous β-catenin expression. We also found that HONE1 hybrid cells have stem cell-like properties, including spheroid formation, up-regulation of CD24+ and CD44+ populations and expression of various embryonic markers. Additionally, chromosome 3 transfer induced epithelial-mesenchymal transformation (EMT) events in HONE1 cells that caused different expression of adhesion molecules and up-regulation of their regulators and other markers such as Twist, Snail, Slug, ZEB1, Sip1, and E-cadherin. As expected, we detected that p53, Rb1, and Smad2 involved pathways were activated in HONE1 hybrid cells, suggesting that physiological β-catenin signaling, via multiple signaling pathways, regulates both pluripotency networks and EMT events.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 420. doi:1538-7445.AM2012-420
Abstract 421: Deciphering the molecular mechanisms behind Oct4(hi) tumor-initiating breast cancer cells with global gene expression
Authors: Bliss Sarah A. , Patel Shyam A. , Rameshwar Pranela
Published: August 2012

Breast cancer continues to have one of the highest rates of incidence despite advances in detection and treatment. One of the most troubling aspects of the disease is its tendency to recur after apparent curative therapy. The ability of a subset of breast cancer cells to remain undetected for years in the patient only to recur years later is of particular interest to our laboratory. Breast cancer is well known to metastasize to the bone marrow where we believe a favorable niche exists for it to remain dormant. Our lab has shown that a subset of breast cancer cells with increased expression of octamer-binding transcription factor 4 isoform A (Oct4A) can form gap junctions with bone marrow stroma cells. Gap junction intercellular communication is important for maintaining dormancy in the breast cancer cells. Further characterization of the Oct4(hi) cells found them to be in mostly the G0/G1 phase of cell cycle, have increased stem cell gene expression, formed tumor spheres, could initiate breast cancer when serially passaged in nude BALB/c mice, and showed increased resistance to carboplatin. To understand the functional differences between the Oct4(hi) cancer cells and the non-tumor-initiating cells, we compared the different breast cancer cell subsets for global gene expression, using Affymetrix gene arrays. The initial analyses confirmed the quality of samples with expression levels grouping together in the principle component analysis. Heat map analyses indicated distinct gene expression in which those that were expressed in the tumor-initiating cells were decreased in the non-cancer stem cells. The tumor-initiating cells showed novel genes that are linked to NFκB, which we have shown to be the master regulator of self-renewal in the cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 421. doi:1538-7445.AM2012-421
Abstract 4211: Development of a prognostic gene expression signature for invasive bladder cancer
Authors: Chu In-Sun , Leem Sun-Hee
Published: August 2012

Bladder cancer comprises of highly heterogeneous tumors and is a malignancy requiring a high surveillance because of the frequent recurrences and the poor clinical outcome when tumors progress into invasive disease. Patients also with superficial and invasive tumors have remarkably different 5-year survival rates reflecting their biological differences. The profiles of gene-expression signatures have become an important and promising way for cancer prognosis and treatment. In addition to their application in cancer class prediction and discovery, microarray gene expression signatures can be used for the prediction of patient survival. Gene expression data were collected from tumor specimens from 165 patients with Korean bladder cancer. We selected then genes use Cox proportional hazard model whose expression patterns are proportionally associated the length of cancer-specific survival (CFS). The expression levels of 256 probes were correlated with CFS time (p<0.005). In poor prognosis subtype, for example, the expression of STK6 (Aurora kinase A), AURKB (Aurora kinase B), and TPX2 (regulator of Aurora kinases) are all up-regulated. One of the best characterized phenomenon of invasion is Epithelial-to-Mesenchymal Transition (EMT). We can't see either vimentin nor Krt19 over-expressed in better prognosis subtype.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4211. doi:1538-7445.AM2012-4211
Abstract 4212: Characterization of the gene expression network that underlies cancer cell-platelet interactions and cancer progression
Authors: Tan Bertrand , Liu Hsuan , Wu Wen-Hsin
Published: August 2012

Understanding how cancer cells outgrow and escape from the primary site is important for development of novel therapeutic scheme. Our recent results from microarray-based gene expression profiling analysis demonstrate differential expression of a cohort of genes (∼220) in the circulating tumor cells, many of which play a role in haemostasis and thrombosis. To screen for putative transcription factors that might contribute to the metastasis-associated gene expression alterations (and possibly the upregulation of haemostatis-associated genes), we first undertook bioinformatics approach by analyzing differentially expressed genes using GeneGo. From the list of potential transcription factors that are identified from the bioinformatics approach, we selected two TFs (CREB and Sp1) for further studies. Consistent with the upregulation of selected genes, ChIP analyses demonstrated promoter occupancy by these two TFs, and further a greater degree of binding in the circulating/metastatic tumor cells. These results suggest their link to the transcriptional induction of the target genes in these cells. Interestingly, we also discovered that the levels of c-fos and Ser15 phosphorylation on p53 underwent an increase in these cells. To dissect the signaling pathways that may underlie the observed transcriptomic alteration, we again applied gene ontology analysis to pinpoint possible signaling networks. Such profiling demonstrated an elevation of the TGFβ2/BMP pathway and a downregulation of EGFR/ErbB signaling in circulating cells compared with the parental line, possibly suggesting a role of the corresponding pathways in the transcriptomic as well phenotypic changes in the metastatic cells. In addition, changes in the expression of several signaling cascade intermediates - Akt, Erk, JNK - were also evident based on immunoblotting results. Our preliminary results have revealed the potential involvement of several transcription factors and signaling pathways in our metastasis experimental model. Discussion of their implications and future work is also included.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4212. doi:1538-7445.AM2012-4212
Abstract 422: Nanog promotes PI3K/Akt and mTOR-mediated invasion in head and neck squamous cell carcinoma
Authors: Rahimy Elham , Yi David , Lew Daniel , Abhold Eric , Kiang Alan , Wang-Rodriguez Jessica , Ongkeko Weg
Published: August 2012

Head and neck squamous cell carcinoma (HNSCC) affects nearly 40,000 new patients annually in the United States with approximately 50% of these patients having a poor prognosis. Frequent metastasis of the primary tumor to nearby cervical lymph nodes and local critical structures poses a major problem in the treatment of HNSCC. To date, several molecular mediators of invasion and metastasis in HNSCC have been studied; however, with the emergence of the cancer stem cell hypothesis, other genes normally associated with self-renewal and pluripotency in stem cells would also need to be evaluated for their role in tumor invasion and metastasis. In the present study, we used various functional assays to determine the role of Nanog in HNSCC invasion and metastasis as well as identify the underlying pathway through which Nanog acts to regulate invasion and metastasis. We observed that Nanog overexpression increased invasion and cell migration in two cell lines (UMSCC-10B and UMSCC-22B) while Nanog knockdown using siRNA decreased invasion and cell migration in both cell lines and in a putative HNSCC cancer stem cell culture which expressed high levels of Nanog. Treatment with LY294002 or Rapamycin using doses that did not significantly affect cell viability and proliferation dramatically reversed both invasion and migration conferred by Nanog overexpression, indicating the effect is most likely dependent upon the PI3K/Akt/mTOR pathway. By western blot, we found that Nanog-overexpressing cells expressed higher levels of p-Akt and p-mTOR compared to control cells or Nanog-knockdown cells, confirming regulation of Akt by Nanog. In addition, Nanog overexpression resulted in the induction of MMP-2/MMP-9 as well as EMT genes Snail, Vimentin, and Twist. This enhanced expression of EMT was consistent with the observed increased migration as demonstrated by a wound healing assay. Nanog regulation in a mouse model is currently under way to confirm our results in vivo. The results of our study support the possibility of screening for Nanog expression as a prognostic tool. Meanwhile, further efforts aimed at elucidating the signaling pathway between Nanog and Akt may reveal novel treatment targets.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 422. doi:1538-7445.AM2012-422
Abstract 4223: Gamma-tocotrienol and simvastatin target TIC and EMT populations in drug-resistant breast cancer
Authors: Gopalan Archana , Yu Weiping , Sanders Bob G. , Kline Kimberly
Published: August 2012

Background and significance: Breast cancer (BC) is the second leading cause of cancer deaths in women. Despite great advances in treatment of early and advanced BC, relapse with drug resistance and metastasis is the biggest impediment to successful remission. Accumulating evidence supports the emerging concept that cancer stem cells (CSCs, also called tumor initiating cells, TICs), a rare tumor cell population that exhibits self-renewal and differentiation properties, are responsible for tumor relapse and metastasis. Hence, identifying molecular mediators responsible for maintenance of TICs and metastasis in drug resistant breast cancer is important to develop better and successful therapeutic strategies. Model system and methods: Acquired tamoxifen resistant (MCF-7/TamR) and doxorubicin resistant (MCF-7/ADR) cell lines as well as their isogenic parental counterparts MCF-7/S and MCF-7/WT, respectively were used in this study. ALDH activity assay was used for characterization and enrichment of TIC population. Immuno-staining/FACS of CD44+/CD24- was used to determine the metastasis potential in-vitro. Western blot analyses were used to detect signal transduction mediators known to be important for maintenance of TIC and EMT populations. Results: Both drug resistant cell lines expressed a higher percentage of TIC population as detected by ALDH activity and elevated EMT population as detected by CD44+/CD24- and increased EMT marker Vimentin and decreased MET marker E-cadherin in comparison with the isogenic sensitive cell lines. Both drug resistant cell lines uniformly expressed higher levels of pStat-3, c-Myc and CyclinD1 in comparison with their respective parental cell lines. Knockdown of Stat-3 using siRNA and Stat-3 inhibitor reduced TIC populations, reduced EMT populations, and decreased c-Myc and CyclinD1, but did not affect Vimentin and E-cadherin protein expression. γT3 and SVA alone and in combination reduced TIC populations as measured by reduced ALDH population and inhibition of mammospheres. Both agents alone and in combination reduced the EMT populations and reduced the levels of p-Stat3, c-Myc and CyclinD1expressed in drug-resistant breast cancer cell lines. Conclusion: These in vitro data demonstrate that drug resistant breast cancer cell lines contain high levels of TIC and EMT sub-populations that are in part controlled by the Stat-3 pathway. Furthermore, αT3 and SVA alone and in combination show the ability to target TIC population and metastasis associated mediators. Supported by the Clayton Foundation for Research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4223. doi:1538-7445.AM2012-4223
Abstract 4224: MiR-7 and its novel target regulate sensitivity to microtubule stabilizing agents
Authors: Liu Lingling , Rogler Charles E. , Horwitz Susan Band , McDaid Hayley M.
Published: August 2012

Microtubule stabilizing drugs, such as Taxol that is used in the treatment of numerous malignancies, cause distinct responses in cancer cells, including apoptosis, mitotic catastrophe, necrosis and accelerated senescence. However, drug resistance, a complex and mutlifactorial phenotype, is a major obstacle to therapy and the foremost cause of disease progression and cancer-related mortality. MiRNAs are small non-coding RNAs that regulate protein expression by targeting their 3′UTRs, and are emerging as key regulators of cancer pathogenesis that can influence sensitivity to chemotherapy and radiotherapy. We have identified miR-7 as a regulator of sensitivity to the microtubule-stabilizing agents, discodermolide and Taxol. Over-expression of miR-7 in the lung cancer cell lines A549 and H2122, strongly inhibited cell proliferation and sensitized the cells to drug-induced apoptosis. Over-expression of miR-7 also increased the number of acidic lysosomes, as measured by β-galactosidase activity, which likely reflects lysosomal stress. It also increased basal and drug-induced caspase activation, which most likely mediate the sensitizing effects of discodermolide and Taxol. In addition, we validated that the transcription factor, Kruppel like factor 4 (KLF4) is a target of miR-7, and that its expression also regulates sensitivity to discodermolide and Taxol. KLF4 is suppressed in discodermolide- and Taxol-treated A549 cells that have a strong senescent phenotype, and is over-expressed in A549 cells that are resistant to either drugs. ShRNA-mediated knockdown of KLF4 in A549 and H2122 also sensitized cells to discodermolide and Taxol though to a lesser degree than miR-7 overexpression, and also inhibited cell proliferation and increased lysosomal β-galactosidase activity and caspase activity. Thus, we hypothesize that suppression of KLF4 partially mediates the potent anti-tumor effects of miR-7 in A549 and H2122 lung cancer cells. In summary, we have identified miR-7 and its novel target, KLF4, as important regulators of sensitivity to discodermolide and Taxol, and suggest that miR-7 is a promising candidate for therapeutic development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4224. doi:1538-7445.AM2012-4224
Abstract 4233: The protein ps20 increases resistance to docetaxel in PC3 and LNCAP prostate cancer cell lines
Authors: Galustian Christine , Hickman Oliver , Smith Richard A.G. , Dalgleish Angus G. , Ukimura Osamu , Gill Indebir S. , Vyakarnam Annapurna , Dasgupta Prokar
Published: August 2012

Introduction The protein ps20 encoded by the WFDC1 gene, is a member of a protein family containing conserved whey-4-disulphide-core (WFDC) domains. Ps20 is upregulated in prostate cancer epithelium and expression is thought to correlate with disease progression. The aim of this study was to determine whether ps20 expressed by prostate cancer cells also increases docetaxel resistance. Methods The cell-lines LNCaP and PC3 express low endogenous ps20, but were transfected with empty vector (EV) or WFDC1 gene vectors that increase WFDC1 RNA and ps20 protein expression by over 100% as measured by qPCR and ELISA. Docetaxel was added from 0.001nM –1µM with or without ps20 antibody (1G7) and cell-killing was assessed by MTT after 48 hours. Results EV-transfected PC3 and LNCaP cells have docetaxel IC50s of 0.223+/-0.05 and 0.16+/- 0.09nM respectively. In WFDC1-transfected PC3 and LNCAP, IC50s are increased to 0.677+/-0.11 and 0.326+/-0.05nM respectively (p<0.001 by paired t-test). Therefore there is a 303% and 203% increase in docetaxel IC50s in PC3 and LNCaP respectively. However, when WFDC1-cells are incubated with docetaxel and ps20 antibody, IC50s are lower than those with EV cells (0.04+/-0.03 and 0.07+/-0.02nM for PC3 and LNCaP respectively, p<0.01 by paired t-test). Therefore, endogenous WFDC1/ps20 on PC3 and LNCaP cells may contribute to docetaxel resistance. Conclusions Ps20 expression by prostate cancer cells is associated with increased resistance to docetaxel- a gold-standard chemotherapy for castrate-resistant prostate cancer. Inhibition of WFDC1/ps20 expression on prostate cancer cells may therefore represent one mechanism of restoring sensitivity in patients that are refractory to docetaxel.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4233. doi:1538-7445.AM2012-4233
Abstract 4237: Inhibition of prostate cancer associated kinases by Tillandsia recurvata: A Jamaican medicinal plant
Authors: Lowe henry , Toyang Ngeh J. , Bryant Joseph
Published: August 2012

Prostate cancer is the second most common cause of death from cancer in men of all ages. The three conventional treatment options include surgery, radiation and chemotherapy. In many cases, these treatments are used in combination. Chemotherapy is however the most widely used tool in prostate cancer treatment especially where the cancer has metastasized or spread to other organs of the body. Increased resistance to current chemotherapies by prostate cancer calls for an urgent need to discover and develop new therapeutics that can slow the growth of cancer cells while having lesser side effects on the patients. The development of target therapies for the treatment of human cancers is revolutionizing the concept of cancer treatment today. This type of therapeutic approach is directed to the inhibition of molecular targets that play a pivotal role in tumor progression such as the kinases. It is only in recent years that kinases have emerged as major targets for therapeutics, particularly for cancer related therapies. Relatively few kinase inhibitors have been identified to date and efforts are being made to discover new small molecules kinase inhibitors. The Island of Jamaica is known for its rich biodiversity and abundance of medicinal plants used in folk medicines. In an effort to discover new anticancer drugs from natural products several Jamaican medicinal plants were screened for anticancer activity. The Jamaican Ball Moss (Tillandsia recurvata L.) was one such plant that exhibited potent activity against the PC-3 prostate cancer cell line in vitro (IC50 = 2.4µg/ml) and was selected for further studies. The crude extract of Ball Moss was screened for interaction with 451 kinases. The crude Ball Moss extract showed potent kinase inhibitory activity against 5 kinases (kd = 8-14 µg/ml), of which 4 are implicated in prostate cancer growth and proliferation. The four kinases are; CSNK2A2, DRAK1, GAK and MEK5. The above results validate the folk use of this medicinal plant as well as make it a good candidate for development into a prostate cancer treatment or for the discovery of the bioactive molecules in the plant. Studies are currently underway to confirm the ability of Ball Moss to halt the growth of prostate tumors in mice as well as measure the inhibitory effects of the crude extract on the 4 related kinases in vivo. The isolation and characterization of the kinase inhibitors in ball moss is also in progress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4237. doi:1538-7445.AM2012-4237
Abstract 4240: The translocator protein 18kDa in the morphogenesis of breast epithelial cells in 3D culture
Authors: Wu Xiaoting , Gallo Kathleen
Published: August 2012

Translocator protein (TSPO) is an 18 kDa mitochondrial membrane protein, first identified as a second binding site for benzodiazepine. Elevated levels of TSPO have been associated with breast cancer progression and with increased proliferation in cancer cells whereas TSPO ligands, PK11195 and Ro5-4864, which may serve as TSPO antagonists, inhibit proliferation in multiple cancer cell lines. Whether TSPO actually contributes to breast cancer progression is not known. In this study, the impact of TSPO up-regulation in breast epithelial cells is being assessed using a 3D Matrigel culture system. MCF10A mammary epithelial cells form well-polarized acini structures with a hollow lumen when grown in 3D Matrigel, whereas expression of oncogenes in MCF10A cells often induces phenotypes such as increased acinar size and filling of the lumen. In this study, a pool of stable TSPO expressing breast epithelial MCF-10A cells has been constructed. TSPO overexpressing MCF-10A cells when grown in 3D matrigel formed larger acini compared with control acini; and displayed abnormal lumen formation. Such a disruption of normal mammary epithelial morphogenesis by TSPO may resemble an early stage breast lesion leading to breast cancer. Immunofluorescent staining revealed increased levels of Ki-67 (a proliferative marker) in TSPO overexpressing acini compared with control acini, suggesting that TSPO promoted proliferation during mammary epithelial morphogenesis. TSPO ligands, including PK11195 and RO5-4864, inhibited proliferation of stable TSPO over-expressing MCF-10A cells in 2D plastic culture. Studies on the impact of TSPO ligands on the morphogenesis of MCF10A and breast cancer cells in 3D culture are being conducted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4240. doi:1538-7445.AM2012-4240
Abstract 4243: Oncogenic K-Ras transforms three-dimensional colon epithelia through upregulation of ERK-MAPK signaling
Authors: Magudia Kirti , Hall Alan
Published: August 2012

Colorectal cancers have the third highest incidence and are the second most common cause of cancer-related death when considering tumors that affect both men and women in the United States. Interestingly, a third of these tumors harbor activating mutations in K-Ras and are associated with worse outcomes. Caco-2 cells, derived from a colorectal carcinoma, are noninvasive and do not harbor mutations in K-Ras, B-Raf or PI3KCA. This cell line has historically been used as a model to study normal, differentiated colonic epithelium in two-dimensional cell culture. Recent studies have used Caco-2 cells in a three-dimensional cell culture system to better model epithelial morphogenesis. In this study, we studied the effects of introducing oncogenic K-Ras into three-dimensionally cultured Caco-2 cells. Control Caco-2 cells seeded in extracellular matrix generate hollow, polarized structures after ten days. In contrast, Caco-2 cells expressing oncogenic K-Ras formed much larger and filled structures that lack a central lumen. Furthermore, western blot analysis of protein lysates made from day 1 and day 10 control structures revealed a strong decrease in cyclin B, suggesting a reduction in the proportion of cells undergoing mitosis. K-Ras structures, however, maintained cyclin B levels, providing evidence of prolonged proliferation. In addition, immunofluorescence of the apical marker aPKC and the tight junction protein ZO-1 revealed that K-Ras structures have disrupted polarity. Caco-2 cells expressing oncogenic B-Raf phenocopied our results with oncogenic K-Ras. Conversely, MEK inhibition with U0126 reverted K-Ras structures back to normal morphology, indicating that K-Ras acts through the ERK-MAPK signaling pathway. The expression of active versions of PI3KCA and RalGDS, other Ras effectors, in Caco-2 cells produced structures identical to control. Further work aims to elucidate a more specific mechanism of Ras downstream of the ERK-MAPK signaling pathway in our three-dimensionally cultured Caco-2 system.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4243. doi:1538-7445.AM2012-4243
Abstract 4244: The role of hypoxia on the tumor-stromal interaction in pancreatic ductal adenocarcinoma
Authors: Heuchel Rainer L. , Longati Paola , Jia Xiaohui , Wagman Annika , Jesenofsky Ralf , Witt Michael R. , Löhr Matthias
Published: August 2012

INTRODUCTION: Pancreatic cancer is characterized by extraordinary fibrosis and pancreatic stellate cells (PSCs) are the major player in synthesizing extracellular matrix (ECM) upon activation. The interaction/communication between PSCs and pancreatic ductal adenocarcinoma (PDAC) cells is essential for inducing stroma/ECM production, pancreatic cancer progression and general therapy resistance. Compared to traditional 2D cell culture, cells in a three-dimensional context demonstrate different behavior and functions as reflected by building up a microenvironment that more closely mimics the one observed in the native tissue. This feature is especially critical for testing drug efficiency as cellular response to drugs is profoundly affected by environmental cues. AIMS & METHODS: Our aim was to build up a more reliable and predictive drug screening system. To this end we set up and compared 2D and 3D cell cultures containing either human PSC and PDAC cells alone or in combination. RESULTS: Among PDAC cells, panc-1 cells are the most commonly used which are also able to form spheres in vitro. We generated mono-spheres from panc-1 cells and compared them to conventional 2D culture. The efficiency of different drugs on viability of cells in panc-1 sphere resulted to be much lower than on viability in 2D cultures. Cells in 3D compared to the 2D culture show higher gene expression for ECM proteins as collagen and fibronectin, for growth factors as PDGF and VEGF, as well as for protein like Cox2, Glut1. We co-cultured panc-1 and PSCs, allowing them to interact and form hetero-spheres in which PSCs localized in the cortical area and were identified e.g. by expression of asma, a marker for PSCs activation. These avascular mini-tumors were further characterized at different levels. CONCLUSION: 3D cultures are more resistant to chemotherapy compared to their 2D counterpart offering a more predictive platform for drug screening before proceeding to more expensive in vivo models and clinical trials. The avascular mini-tumors can be considered as an alternative in vitro cell model for studying pancreatic cancer development and fibrosis from a more complete perspective.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4244. doi:1538-7445.AM2012-4244
Abstract 4371: RRx-001 modulates intratumor blood flow in SCCVII and U87 tumors
Authors: Scicinski Jan , Oronsky Bryan , Ning Shoucheng , Minchinton Andrew , Knox Susan
Published: August 2012

The tumor-specific provascular effects of the anti-cancer agent RRx-001, a novel nitrogen oxide donor and allosteric hemoglobin modifier in Phase 1 clinical trials were investigated using contrast enhanced ultrasound (CEUS) and whole tissue section quantitative immunohistological staining. Changes in tumor blood flow by CEUS in SCCVII tumor bearing mice following RRx-001 (3, 6, 12 mg/kg) were dose and time dependent, reaching maximum perfusion 6 h after treatment, returning to baseline within 72 h. Microregional effects of RRx-001 (15 mg/kg) on tumor blood flow and oxygenation by immunohistological staining were studied in mouse SCCVII and human U87 tumors. SCCVII tumors exhibited regions of intermittent perfusion exemplified by co-localization of vessels with the hypoxia marker pimonidazole commonly occurring throughout the tissue. Only 21% of vessels showed perfusion after a bolus dose of the perivascular stain DiOC7. This increased to 28% 12 h post RRx-001. Paradoxically, an increase in tissue oxygenation was not seen while modestly increased pimonidazole binding was observed. However, preliminary radiobiological assessment suggested tumors were radiosensitized when irradiated 10 min after 12 mg/kg RRx-001. In the U87 tumor a decrease in vessel perfusion at 90 min post RRx-001 administration was observed. Blood flow over large areas of treated tumors was completely shut down. These areas stained positive for CD31 and tissue did not appear necrotic. Analysis indicated 25% reduction in the fraction of perfused vessels and un-perfused tissue by area at 90 min returning to near normal levels at 12 h. There was no significant increase in the fraction of necrosis at 12 h suggesting that the large un-perfused regions seen at 90 min had recovered rather than become necrotic. No significant change in tumor hypoxia was seen at 90 min or 12 h. For both tumor types, RRx-001 appeared to cause the loss of perfusion in large regions of the tumor however, at the 12 h time point, both tumor types showed increase in vessel perfusion but no significant decrease in hypoxia. These data suggest a redistribution of blood flow in both tumor types while differences between the tumors were related to tumor architecture and distribution of α-SMA. RRx-001 shows promise for short-term blood flow redistribution in tumors with a pericyte-rich vasculature with contractile proteins such as α-SMA. Expression of α-SMA in tumor vasculature could therefore be useful for predicting tumor response to RRx-001.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4371. doi:1538-7445.AM2012-4371