bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3463: Identification and characterization of novel cancer cell derived factors that dictate vascular endothelial cell biology
Authors: Walshe Tony , Bourdelais Justin J. , Lee Arleide , Ouro-Djobo Rakib , Vishnudas Vivek , Kiebish Michael , Sarangarajan Rangaprasad , Narain Niven R.
Published: May 2015

Cell-cell communication in the tumor microenvironment drives tumor growth and facilitates neovessel recruitment from the adjacent vasculature. The aim of these experiments were to identify novel cancer derived factors that modulate the vascular endothelial response in a paracrine manner. An array of cancer cell lines from multiple tissue types (Panc1; Hep2G; LnCap; Skmel; Hep3B; PL45; SCC25; CaCo2; PaCa2; SKBR3) were grown under normoxic or hypoxic (1% oxygen) conditions for a total of 20 conditions. After 48 hours, conditioned media was collected and filtered prior to testing on endothelial cells. The effects of these media on endothelial apoptosis, proliferation and tube formation were investigated. In tandem with these endothelial cell fate assays, the proteomic and lipidomic profile of these 20 conditions were profiled and assessed using the Berg Interrogative Biology™ platform. Our results reveal that conditioned media from all cancer cells tested can alter endothelial cell apoptosis, proliferation rate and tube formation in 3D matrigel assays i.e. critical cell fate decisions that enable formation of nascent vessel formation. Interestingly, the predominant effect of tumor-derived conditioned media on endothelial apoptosis was a protective anti-apoptotic effect, with the discernable exception of normoxic Panc1 derived media. An unexpected finding was the general anti-proliferative effects of tumor-derived conditioned media on endothelial cell growth, with the exception of Hep2G, LnCap and PaCa2 cell derived media. Using a media fractionation strategy and the Berg Interrogative Biology™ platform, we identified several endothelial anti-apoptotic candidates from Panc1 cells within a unique molecular weight range. Upon application of ASO gene knockdown in Panc1 cells, these targets were verified for apoptotic and proliferation effects on vascular endothelial cells.Specific factors that determine paracrine cell-cell interaction in the tumor microenvironment have been identified and validated using the Berg Interrogative Biology™ platform.

Citation Format: Tony Walshe, Justin J. Bourdelais, Arleide Lee, Rakib Ouro-Djobo, Vivek Vishnudas, Michael Kiebish, Rangaprasad Sarangarajan, Niven R. Narain. Identification and characterization of novel cancer cell derived factors that dictate vascular endothelial cell biology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3463. doi:10.1158/1538-7445.AM2014-3463
Abstract 3466: Mediated by cancer cell and stroma cell TGF-β signaling, the periostin / β-Ig-H3 expression ratio is altered during breast cancer progression
Authors: Coleman Michelle M. , Burman Anindita Das , Lance Amanda L. , Dreau Didier
Published: May 2015

Involved in the maintenance of normal breast tissue, the extracellular matrix (ECM) also supports the progression of several human cancers. In particular, both periostin and TGF-β-induced (β-Ig-H3) whose expression is regulated by transforming growth factor-β (TGF-β) signaling, are differentially expressed in many human cancers. While a link between increased periostin expression and poor breast cancer outcomes has been shown, our recent data suggest that overall periostin expression is variable and is poorly correlated with human breast cancer progression. Therefore, in this study we investigated the ratio between periostin and β-Ig-H3 expression and the relationship between TGF-β signaling and expression of these ECM proteins by cancer cells and stromal cells during human breast cancer progression. Expressions of periostin and β-Ig-H3 were determined in biopsy specimens collected from 61 breast cancer patients by immunohistochemistry and correlated with clinico-pathologic features. Additionally, periostin, β-Ig-H3, and TGF-β expression in multiple stroma and tumor cells were evaluated in vitro using western-blots and ELISAs. Increases in the periostin / ß-Ig-H3 ratio correlated with increased the AJCC stage and progesterone positive status of the breast cancer specimens tested (p<0.05). In vitro, murine L929 fibroblasts and 4T1 mammary cancer cells secreted higher periostin concentrations compared to J774 and RAW macrophages (p<0.05). Furthermore, the periostin / ß-Ig-H3 protein expression ratio of 4T1 cells was higher than those of J774 and RAW cells. Following treatment with TGF-ß that activated the SMAD pathway, L929 and 4T1 cells expressed higher periostin concentrations (p<0.05). In contrast, the high ß-Ig-H3 secretion by L929 fibroblasts and J774 macrophages remained unchanged following TGFß treatment (n.s.). These observations support a role for TGF-ß and periostin signaling loops between stroma and cancer cells in the microenvironment and may indicate a role of the periostin / ß-Ig-H3 ratio as a predictor of breast cancer aggressiveness.

[This works was supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007)]

Citation Format: Michelle M. Coleman, Anindita Das Burman, Amanda L. Lance, Didier Dreau. Mediated by cancer cell and stroma cell TGF-β signaling, the periostin / β-Ig-H3 expression ratio is altered during breast cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2014-3466
Abstract 3467: Progesterone inhibits endometrial cancer growth and invasiveness by modulating the TGF-ß pathway
Authors: Bokhari Amber A. , Lee Laura R. , Raboteau Dewayne , Hamilton Chad A. , Maxwell George L. , Turbov Jane M. , Thaete Larry G. , Rodriguez Gustavo C. , Syed Viqar
Published: May 2015

Objectives: Our group has shown previously that progesterone may exert a chemoprotective effect against endometrial cancer through modulation of TGF-β signaling and concomitant activation of apoptosis in the endometrium. In addition, it has been shown that increased expression of TGF-β isoforms in human endometrial cancer correlates with decreased survival and poor prognosis. These data suggest that the TGF-β pathway may be an attractive target for chemoprevention strategies. The goal of this study was to further characterize the effect of progesterone on TGF-β signaling pathway components (TGF-β isoform and Smads) and on TGF- β-induced pro-tumorigenic activities in endometrial cancer cell lines.

Methods: The expression levels of TGF-β ligands (TGF-β1, TGF-β2 and TGF-β3), TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3) and SMADs (pSMAD2/3, SMAD2/3 and SMAD-4) were determined by immunoblotting in HEC-1B endometrial cancer cells exposed to progesterone for 24, 72 and 120 h. Proliferation and cellular invasion assays were used to access the functional effects of progestin exposure in the HEC-1B and Ishikawa endometrial cancer cell lines.

Results: A marked decrease in TGF-β1and TGF-β3 expression was observed at 72 h after treatment with progesterone. Expression of TGF-βR1, TGF-βR2, SMAD2/3 and pSMAD2/3 were substantially reduced at 72 h while levels of SMAD4 and TGF-β2 expression were reduced at 120 h following progesterone. TGF-βR3 expression levels were not affected by any treatment at any time point. Furthermore, TGF-β1-induced cancer cell proliferation and invasion was effectively inhibited by progesterone. Cellular proliferation and invasion were significantly reduced by progestin compared to controls even in the presence of exogenous TGF-β.

Conclusions: These results suggest that the down-regulation of TGF-β signaling may be a key mechanism underlying progestin inhibition of endometrial carcinogenesis.

Citation Format: Amber A. Bokhari, Laura R. Lee, Dewayne Raboteau, Chad A. Hamilton, George L. Maxwell, Jane M. Turbov, Larry G. Thaete, Gustavo C. Rodriguez, Viqar Syed. Progesterone inhibits endometrial cancer growth and invasiveness by modulating the TGF-ß pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2014-3467
Abstract 3471: Gene expression profiling of a single laser capture microdissected (LCM) cell
Authors: Chu Shirley
Published: May 2015

Laser Capture Microdissection (LCM) is a technique providing a rapid and reliable method to procure purified cell populations, either as multiple single cells or a group of cells, from a heterogenic tissue sample allowing a targeted approach to genomic profiling. When analyzing gene expression profiles from a group of cells, the average profile may not be a true representation of the many different profiles that could exist even in a pure cell population (e.g., in different states of growth, differentiation, or activation). As a result, the transcriptional variability of individual cells and any insight into the relationship between specific genes in single cells are lost. To fully understand the complexity of tissue and cellular heterogeneity, it is necessary to measure molecular signatures at the single cell resolution. Here we developed a workflow for gene expression profiling of a single LCM cell that can also be applied to small number of LCM cells. The ArcturusXT™ LCM system was used to harvest single cells from frozen human tumor tissue sample which were then processed for quantitative RT-PCR (qRT-PCR) gene expression analysis.

Citation Format: Shirley Chu. Gene expression profiling of a single laser capture microdissected (LCM) cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3471. doi:10.1158/1538-7445.AM2014-3471
Abstract 3473: Identification of progesterone-dependent genes regulated by ablation of mitogen-inducible gene-6 (Mig-6) in the murine uterus
Authors: Kim Tae Hoon , Yoo Jung-Yoon , Jeong Jae-Wook
Published: May 2015

Mitogen-inducible gene-6 (Mig-6) is an important mediator of progesterone signaling to inhibit estrogen signaling in the uterus. Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. Although Mig-6 is an important tumor suppressor in endometrial cancer, relatively little is known about the molecular mechanisms of Mig-6 in endometrial cancer. To identify the molecular pathways regulated by Mig-6 in a steroid-hormone dependent manner, we performed microarray analysis on the uterus of ovariectomized PRcre/+Mig-6f/f (Mig-6d/d) mice treated with progesterone for 6 hours. In the analysis of GeneChip® Mouse Genome 430 2.0 Array, 2,034 genes, 1,062 genes were up-regulated and 972 genes were down-regulated, were altered by P4 treatment. To determine which pathways are regulated by P4 treatment, we performed pathway analysis using Ingenuity Systems Software. Genes that involved in Estrogen Receptor, IGF-1, PI3K/AKT, JAK/Stat, ERK/MAPK, ILK, p53, PTEN, and Wnt/β-catenin signaling were found to be P4 response. In addition, genes that regulated the molecular mechanisms of cancer were disproportionated. And in microarray, 2,236 genes, 1,883 genes were up-regulated and 356 genes were down-regulated, were regulated by Mig-6 ablation on P4 treatment. Genes involved in ILK, p53, PTEN, PI3K/AKT, and Wnt/β-catenin signaling were found to be deregulated upon Mig-6 ablation on P4 response. Furthermore 324 genes of the 2,034 genes were regulated by P4 treatment were dys-regulated in Mig-6d/d mice treated with P4 compared to Mig-6f/f control mice. These results showed that gene expression pattern was altered by the deletion of Mig-6 and the change of these genes was dependent on P4 in uterus. Thus the further investigation should be completed to identify molecules associated with Mig-6 and the identification of relation between these responsive genes will contribute to define the molecular pathways regulated by Mig-6 in uterine biology.

(This work was supported by NIH, R01 HD057873 and American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.)

Citation Format: Tae Hoon Kim, Jung-Yoon Yoo, Jae-Wook Jeong. Identification of progesterone-dependent genes regulated by ablation of mitogen-inducible gene-6 (Mig-6) in the murine uterus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3473. doi:10.1158/1538-7445.AM2014-3473
Abstract 3482: A robust TALENs system for highly efficient mammalian genome editing
Authors: Zhang Siliang , Feng Yuanxi , Huang Xin
Published: May 2015

Recently, transcription activator-like effector nucleases (TALENs) have emerged as a highly effective tool for genomic editing. A pair of TALENs binds to two DNA recognition sites separated by a spacer sequence, and the dimerized FokI nucleases at the C terminal then cleave DNA in the spacer. Because of its modular design and capacity to precisely target almost any desired genomic locus, TALENs is a technology that can revolutionize the entire biomedical research field. Currently, for genomic editing in cultured cells, two plasmids encoding a pair of TALENs are co-transfected, followed by limited dilution to isolate cell colonies with the intended genomic manipulation. However, uncertain transfection efficiency becomes a bottleneck, especially in hard-to-transfect cells, reducing the overall efficiency of genome editing. We have developed a robust TALENs system in which each TALEN plasmid also encodes a fluorescence protein. Thus, cells transfected with both TALEN plasmids, a prerequisite for genomic editing, can be isolated by fluorescence-activated cell sorting. Our improved TALENs system can be applied to all cultured cells to achieve highly efficient genomic editing. Furthermore, an optimized procedure for genomic editing using TALENs is also presented. We expect our system to be widely adapted by the scientific community.

Citation Format: Siliang Zhang, Yuanxi Feng, Xin Huang. A robust TALENs system for highly efficient mammalian genome editing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3482. doi:10.1158/1538-7445.AM2014-3482
Abstract 350: Virtualization of drug testing by predictive systems biology modeling for optimal drug treatment of cancer cells and drug repositioning
Authors: Kuehn Alexander , Dreher Felix , Peycheva Svetlana , Yildiriman Reha , Lehmann Verena , Kessler Thomas , Wierling Christoph , Lehrach Hans , Lange Bodo M.
Published: May 2015

Traditionally, tumors have been classified based on their tissue of origin and their histopathological characteristics and consequently patients are divided into groups where they are treated identically despite substantial differences in genetic profiles. Identification of genes causally implicated in cancer development has led to the development of ‘stratified’ medicine, which adjusts a patient’s therapy based on biomarkers that identify some of the patients expected not respond to a specific therapy. However, many tumor genome sequencing projects have shown that many more gene mutations drive the development of cancer than previously thought, making every tumor unique. These results in low success rates and thereby high cost in drug approval as the identified drugs are only effective for particular patient groups while large patient cohorts have no clear clinical benefit. In the early process of drug development, drugs are therefore screened on large cancer cell line collections to define drug applicability and to determine potential tumor targets.

Here, we report the results of a computational modeling platform, ModCell, allowing the prediction of individual drug effects using large-scale genomic and transcriptomic data to virtualize such cell line screening. We validated this approach two-fold: on publicly available data from the cancer cell line encyclopedia, that comprises pharmacological profiles for anti-cancer drugs across a library of cancer cell lines as well as on cancer cell line culture experiments. We provide evidence that ModCell is able to reproducibly predict the effects of individual drugs with a confounding 80% accuracy, to predict combinatory drug action and to identify new applications for existing drugs. Thus, computational modeling using ModCell can improve todays drug development by accelerating and partly replacing work which would have otherwise be conducted in the laboratory and in the clinic.

Citation Format: Alexander Kuehn, Felix Dreher, Svetlana Peycheva, Reha Yildiriman, Verena Lehmann, Thomas Kessler, Christoph Wierling, Hans Lehrach, Bodo MH Lange. Virtualization of drug testing by predictive systems biology modeling for optimal drug treatment of cancer cells and drug repositioning. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 350. doi:10.1158/1538-7445.AM2014-350
Abstract 3509: Overexpression of 14-3-3γ contributes to chromosomal instability in human lung cancer
Authors: Gomes Cecil J. , Centuori Sara , Martinez Jesse D.
Published: May 2015

Lung cancer is the most common cause of cancer-related deaths, and nearly all lung cancers exhibit genomic instability which results in an abnormal number of chromosomes known as aneuploidy. In many lung cancers, 14-3-3 proteins have been shown to be aberrantly activated or suppressed, which implies that these proteins have potential roles in tumorigenesis. Our lab has demonstrated that over expression of one 14-3-3 family member, 14-3-3γ, in a non-small cell lung cancer cell line leads to an increase in the number of cells that exhibit polyploidy, suggesting that this 14-3-3 protein can disrupt normal chromosome segregation. Additionally, overexpression of 14-3-3γ leads to atypical DNA replication and cell cycle progression. Further evaluation of the polyploid population induced by overexpressing 14-3-3γ revealed that a majority of the cells were multinucleated, often with two or more nuclei of unequal size and morphology. Live cell microscopy shows that this nonlinear marked increase in nuclear content is due to unequal distribution of DNA to the daughter cells upon cellular division, promoting chromosomal instability. Our data indicates that 14-3-3γ may play an important role in maintaining normal diploidy, but when overexpressed, as seen in many lung cancers, can lead to increased levels of chromosomal instability and progression into aneuploidy.

Citation Format: Cecil J. Gomes, Sara Centuori, Jesse D. Martinez. Overexpression of 14-3-3γ contributes to chromosomal instability in human lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2014-3509
Abstract 3510: N-Myc positively influences mitochondrial biogenesis in neuroblastoma
Authors: Sharma Manika , Graves James A.
Published: May 2015

Background:

Neuroblastoma is the most common extracranial tumor of childhood., accounting for approximately 7% of all cancer in children. The morbidity and mortality are affected by a number of factors - perhaps most significantly by amplification of the oncogene, N-MYC. Advanced stage disease with N-MYC overexpression confers the highest mortality. The N-MYC gene product (N-Myc) regulates the transcription of many genes, and when overexpressed is thought to enhance the cell cycle and dysregulate apoptosis. The function of N-Myc is virtually identical to another oncoprotein of the same family, c-Myc, yet is restricted by developmental stage and cell type. Overexpression of c-Myc has been shown to increase mitochondrial biogenesis by alterations in mitochondrial dynamics. It has been proposed that these changes may contribute to c-Myc induced tumorigenesis. We hypothesize that N-Myc amplification will have similar effects on mitochondrial biogenesis in neuroblastoma.

Design/Methods:

To perform these studies we utilized a cell line with endogenous levels of N-Myc (SK-N-SH). In these cells we have ectopically overexpressed N-Myc and examined the cells for changes in the mitochondria with various assays, including: Western blots, electron microscopy, flow cytometry, ATP assay and confocal microscopy.

Results:

We found a X-fold increase in mitochondrial mass in cells that overexpress N-Myc,, as well as a more tubular appearance with less organized cristae. Additionally, N-Myc changed the expression of various mitochondrial fusion and fission proteins. The most dramatic change was seen in the expression of the fusion protein Opa1, which changed by ten-fold. This, combined with a decrease in expression of fission proteins DRP1 and MFF, may account for the more tubular appearance of mitochondria in N-Myc overexpressing cells.

Conclusions

N-Myc amplification appeared to increase mitochondrial mass. This may occur as a result of increased expression of mitochondrial fusion proteins, as previously observed in c-Myc overexpressing cells. Additionally, mitochondrial function is influenced by N-Myc overexpression. Therefore, N-Myc amplification appears to positively influence mitochondrial biogenesis in neuroblastoma. Further studies are needed to determine if this increase in mitochondrial mass and function is necessary for the development and maintenance of neuroblastoma.

Citation Format: Manika Sharma, James A. Graves. N-Myc positively influences mitochondrial biogenesis in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3510. doi:10.1158/1538-7445.AM2014-3510
Abstract 3515: Increased TSH signaling by overexpressed thyrotrophin receptor is essential for B-RafV600E induced thyroid carcinogenesis
Authors: Kim Young Hwa , Choi Yong Won , Kim Jang-Hee , Park Tae Jun
Published: May 2015

B-RafV600E oncogene mutation occurs most commonly in papillary thyroid cancer (PTC), and is associated with tumor initiation. However, a genetic modification by B-RafV600E in the thyrocytes results in oncogene induced senescence (OIS). In the present study, therefore, we attempted to evaluate the factors involved in the senescence overcome program in PTC. First of all, we observed downregulation of p-Erk1/2 and upregulation of dual specific phosphatase 6 (DUSP6) in the PTC with B-RafV600E mutation. In B-RafV600E expressing thyrocytes, although DUSP6 mRNA was upregulated, its protein and phosphatase activity were similar to those of normal thyrocytes. However, DUSP6 activity was reactivated when ROS was downregulated by thyroid stimulating hormone (TSH). Moreover, upregulation of MnSOD by TSH was observed in thyrocytes and PTC. Although serum TSH was not increased, its receptor was markedly upregulated in PTC with B-RafV600E. TSH together with DUSP6 inhibited B-RafV600E induced senescence in the thyrocytes, and reactivated Ras/Akt/GSK3β resulted in c-Myc protein stabilization by inhibition of its degradation. These observations led us to conclude that increased TSH signaling by overexpression of TSH receptor is essential for B-RafV600E induced papillary thyroid carcinogenesis.

Citation Format: Young Hwa Kim, Yong Won Choi, Jang-Hee Kim, Tae Jun Park. Increased TSH signaling by overexpressed thyrotrophin receptor is essential for B-RafV600E induced thyroid carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3515. doi:10.1158/1538-7445.AM2014-3515
Abstract 3517: Role of primary cilia in breast and prostate cancer
Authors: Hassounah Nadia B. , Nunez Martha , Nagle Raymond , McDermott Kimberly M.
Published: May 2015

Primary cilia are microtubule-based organelles expressed on many mammalian cell types and help sense the extracellular environment. Signaling pathways important in development, tissue homeostasis, as well as cancer are known to be regulated by primary cilia, such as the Wnt and Hedgehog pathways. However, little is known about the role of primary cilia in cancer. We have previously demonstrated in a published study that primary cilia expression was significantly decreased through human prostate cancer progression (PLoS ONE 8 (7), 2013). We also discovered that primary cilia lengths were shorter through prostate cancer progression, which suggests dysfunction. In addition, we observed that primary cilia expression and length correlated with canonical Wnt signaling in normal human prostate tissue, premalignant lesions, and a subset of cancers. This data suggested that primary cilia dysfunction may alter Wnt signaling in the human prostate. We have also characterized primary cilia in human breast cancers, and similarly found a significant loss of primary cilia with breast cancer progression. We therefore hypothesize that primary cilia suppress tumorigenesis in these tissues, and consequently that primary cilia loss promotes cancer formation by altering signaling pathways. We are currently testing this hypothesis by knocking out primary cilia in a tumorigenic mammary mouse model and measuring changes in tumor growth, as well as changes in signaling pathways, like Wnt and Hedgehog signaling. Preliminary data demonstrates enhanced tumor growth in the absence of primary cilia, thus supporting our hypothesis that primary cilia may act as tumor suppressor organelles.

Citation Format: Nadia B. Hassounah, Martha Nunez, Raymond Nagle, Kimberly M. McDermott. Role of primary cilia in breast and prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3517. doi:10.1158/1538-7445.AM2014-3517
Abstract 3526: The mTORC2 component RICTOR plays a key role in lung cancer cell growth
Authors: Cheng Haiying , Zou Yiyu , Borczuk Alain , Qiu Wanglong , Piperdi Bilal , Kim Mimi , Halmos Balazs , Perez-Soler‎ Roman
Published: May 2015

RICTOR (Rapamycin-insensitive companion of mTOR protein) is a key component of mTORC2 (the mammalian target of rapamycin complex 2). One of the most-recognized targets for RICTOR-mTORC2 is AKT (Ser473). RICTOR also carries functions independent of mTORC2. RICTOR signaling has been suggested to play key roles in regulating cancer cell migration, invasion and metastasis in breast, ovarian, colorectal cancers and gliomas. The potential roles of RICTOR in lung cancer remain to be elucidated. We first examined the expression profile of RICTOR in primary lung tumor specimens and in lung cancer cell lines by immunohistochemistry. Among 125 FFPE patients' specimen, ninety nine were stained positive for RICTOR (intensity 1+ to 3+). More interestingly, the RICTOR IHC expression is stronger in squamous cell lung cancer in comparison to adenocarcinoma. Furthermore, twenty four out of 36 lung cancer cell lines showed positive RICTOR IHC staining. Thus, RICTOR is expressed in most lung tumors. To investigate the role of RICTOR in lung cancer cell growth, we found that RICTOR knockdown by siRNA reduced colony formation in A549, HCC827 and Calu3 cells, regardless of KRAS or EGFR mutational status (A549 is KRAS mutated whereas HCC827 is EGFR mutated). Western blot confirmed RICTOR knockdown and also decreased level of its downstream pAKT S473. We further blocked RICTOR signaling by utilizing inducible shRNAs of RICTOR. Similar inhibition of lung cancer cell growth was observed. Moreover, to test the in vivo role of RICTOR, we preformed xenograft mouse experiments. When RICTOR was inducibly knockdown by the presence of doxycycline, the growth of A549 lung tumor xenografts was markedly reduced by 60% (P<0.05). Taken together, our study provides the rational basis for a potential RICTOR-targeted therapy in lung cancer.

Citation Format: Haiying Cheng, Yiyu Zou, Alain Borczuk, Wanglong Qiu, Bilal Piperdi, Mimi Kim, Balazs Halmos, Roman Perez-Soler‎. The mTORC2 component RICTOR plays a key role in lung cancer cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3526. doi:10.1158/1538-7445.AM2014-3526
Abstract 3530: The parathyroid hormone-related peptide region likely features seven discrete breast cancer susceptibility loci
Authors: Freeman Adam N. , Martin Terence ‘Jack’ , Henderson Michael , Makalic Enes , Schmidt Daniel F. , Kapinski Miroslaw K. , Southey Melissa C. , Giles Graham G. , Hopper John L.
Published: May 2015

Parathyroid hormone-related protein (PTHrP), the product of the PTHLH gene, has long been implicated in the mechanism by which breast cancers establish and grow as metastases in bone. Paradoxically, a prospective clinical study clinical suggests that its production early in breast cancer in a cancer is actually protective. Multiple recent Genome-Wide Association Studies (GWAS) have confirmed a single susceptibility locus immediately upstream of the PTHLH gene to be associated with breast cancer. In this study we compare the findings of two GWAS, the ABCFS and the MCCS, along with a large imputed meta-analysis combining the ABCFS, BBCS, DFBBCS, MARIE, SASBAC, HEBCS, GC-HBOC, UK2, BPC3, BCFR and SardiNIA data. To better discern the signals of association with common variant Single Nucleotide Polymorphisms (SNPs) in the PTHLH gene region, we compare results from a conventional marginal analysis performed by plink with those from DepTH, a novel bootstrap permutation-based method that considers multiple contiguous SNPs. These are plotted on the genome and analysed in conjunction with hapmap, encode expression and regulation, genome-wide epistasis, eQTLs, and genetic data. Whereas the classic approach identifies only one SNP (“top hit”), our analysis suggests there are at least 7 discrete regions in and around PTHLH in which there are susceptibility variants, rare or common, with potentially independent functions on disease risk. In addition, exhaustive genome-wide epistasis demonstrates statistical interactions between PTHLH and multiple other gene regions including calcitonin and the vitamin D receptor in breast cancer.

Citation Format: Adam N. Freeman, Terence ‘Jack’ Martin, Michael Henderson, Enes Makalic, Daniel F. Schmidt, Miroslaw K. Kapinski, Melissa C. Southey, Graham G. Giles, John L. Hopper. The parathyroid hormone-related peptide region likely features seven discrete breast cancer susceptibility loci. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2014-3530
Abstract 3534: Epigenetic control of the tumor suppressive microRNA miR-34a in bladder cancer
Authors: Huang Wen-Yu , Chen Pi-Che , Yeh Chia-Ming , Cheng Frank H.C. , Hsieh Hsiao-Yen , Shen Cheng-Huang , Hsu Cheng-Da , Chan Michael W.
Published: May 2015

Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. One of the hypotheses suggests that cancers arise from cancer stem-like cell (CSC) with surface antigen CD44. MicroRNAs (miRNAs) are endogenous, non-protein-coding RNAs that can regulate the expression of their target mRNAs. Up to 90% of human genes can be regulated by this mechanism. However, the role of miRNAs in the regulation of CSC in bladder cancer is not fully explored. Bioinformatics prediction identified that miR-34a regulates the expression of CD44. In this study, we aimed to investigate how miR-34a are regulated in bladder cancer. Expression analysis showed that miR-34a was down-regulated in UMUC3 bladder cancer cells. COBRA assay and bisulphite pyrosequencing demonstrated extensive DNA methylation of miR-34a in this cell. Treatment of DNMT inhibitor, 5-aza restored miR-34a expression in UMUC3 cells thus suggesting that miR-34a is epigenetically silenced in UMUC3 cells. Over-expression of miR-34a resulted in a reduction of chemo-resistance to cisplatin in UMUC3 cells. This may be due to a down-regulation of CD44 and another predicted miR-34a target, c-myc in this cell. Recently, additional mechanism namely, competitive endogenous RNA (ceRNA) hypothesis in controlling the function of miRNAs are demonstrated. To test this hypothesis in bladder cancer, we overexpressed 3′UTR of CD44 in TSGH8301 bladder cancer cells which expressed miR-34a, and resulted in an up regulation of another target of miR-34a, c-myc in this cells. Since amplification of chromosome 8q24 where c-myc resides is frequently observed in bladder cancer, we investigated the relationship between expression of c-myc and CD44 in a publicly available expression array data set (GDS1479) using 60 bladder cancer patients. Interestingly, a positive correlation was found between the expression of c-myc and CD44 (P=0.0016) in this data set. In conclusion, aberrant promoter methylation and ceRNA mechanism may contribute to attenuate the function of miR-34a in bladder cancer. The tumor suppressive role of miR-34a in controlling CSC phenotype in bladder cancer deserves further investigation.

Citation Format: Wen-Yu Huang, Pi-Che Chen, Chia-Ming Yeh, Frank H.C. Cheng, Hsiao-Yen Hsieh, Cheng-Huang Shen, Cheng-Da Hsu, Michael WY Chan. Epigenetic control of the tumor suppressive microRNA miR-34a in bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3534. doi:10.1158/1538-7445.AM2014-3534
Abstract 3541: HOTAIR modulates β-catenin signaling pathway through NLK and NFAT5 in human glioblastoma
Authors: Zhou Xuan , Ren Yu , Han Lei , Kang Chunsheng
Published: May 2015

Background

Glioblastoma (GBM) is the most common malignant tumor of human central nervures system with poor prognosis. A new class of transcripts, long noncoding RNAs (lncRNAs), has been recently found to be pervasively transcribed in the genome. Recent progress suggests that the involvement of lncRNAs in human cancers could be far more prevalent than previously appreciated. HOTAIR, lncRNA Hox transcript antisense intergenic RNA, has been characterized as a novel hall marker to predict poor prognosis in glioma. HOTAIR serves as a modular scaffold by interacting with the polycomb repressive complex 2 (PRC2) and the lysine-specific demethylase 1 (LSD1) corepressor for element-1-silencing transcription factor (CoREST) complex to silence the HOXD loci in trans. Constitutive activation of the Wnt/β-catenin pathway is a common feature of human cancers and contributes to its development, progression and metastasis.

Methods

RNA sequencing and qPCR were used to determine HOTAIR expression status in GBM samples. TOP/FOP flash luciferase assays and western blot were employed to identify the effect of HOTAIR's function on the Wnt/β-catenin pathway activity. In addition, FCM, transwell assay, clone formation assay were used to examine HOTAIR's biological function in regulating GBM cell phenotype.

Results

We show β-catenin pathway is associated to HOTAIR expression by RNA sequencing in GBM cell lines and large scale glioma samples. Besides, increasing NLK and NFAT5 expression, PKM2 and β-catenin pathway inhibition is induced by HOTAIR knockdown in vivo and in vitro. Moreover, HOTAIR knockdown can partially overcome hypoxia stimulation to U87 and U87VIII cells, block cell cycle at G1 phase, and attenuate GBM cell invasion or EMT like transformation. And EZH2 domain may contribute to HOTAIR's biological function.

Conclusion

Our data indicate that HOTAIR expression is correlated with the overexpression ofβ-catenin pathway activation in both GBM cell lines and tissues. And HOTAIR can serve as a therapeutic target in gliobalstoma, relating its oncogenic effects to activation of β-catenin pathway.

Citation Format: Xuan Zhou, Yu Ren, Lei Han, Chunsheng Kang. HOTAIR modulates β-catenin signaling pathway through NLK and NFAT5 in human glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3541. doi:10.1158/1538-7445.AM2014-3541
Abstract 3555: LncRNA-21a is a potential tumor suppressor in breast cancer
Authors: Singh Ramesh , Mo Yin-yuan
Published: May 2015

Long non-coding RNA (lncRNA)-21A is a polymerase III driven non-coding RNA consisting of 324 nucleotides in length. LncRNA-21A was originally identified through in silico analysis and its expression was then confirmed in human cell lines. Further study suggested that lncRNA-21A can control the proliferation of human tumor cell lines, possibly through regulation of CENP-F expression. However, little is known whether lncRNA-21A plays a role in breast cancer. In this study, we reported that lncRNA-21A is downregulated in breast cancer, as detected by RT-PCR analysis of breast cancer tissue scan, suggesting its possible role as a tumor suppressor. In consistent with this finding, lncRNA-21A promotes apoptosis and attenuates cell invasion ability in breast cancer cells. Western blot revealed that overexpression of lncRNA-21A can reduce the phosphorylation of Akt in MCF-7 cells. To determine its underlying mechanism, we performed RNA precipitation assays with the synthetic biotin labeled lncRNA-21A RNA probe followed by mass spectrometry analysis, and we identified hnRNP U and EWS as binding partners for lncRNA-21A. Suppression of EWS by RNAi leads to a reduced level of phosphorylated Akt, suggesting its role in lncRNA-21A-medited Akt activity. Of great interest, we found that like microRNAs, lncRNA-21A can also be secreted through exosomes into conditioned medium. Furthermore, the secreted lncRNA-21A from normal breast epithelial cells can be functionally taken up by recipient metastatic breast cancer cells, suppressing the cell invasion. Together, these results suggest that as a potential tumor suppressor, lncRNA-21A may be used by the host through exosome-mediated gene transfer as a defense mechanism against invasive tumor cells.

Citation Format: Ramesh Singh, Yin-yuan Mo. LncRNA-21a is a potential tumor suppressor in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3555. doi:10.1158/1538-7445.AM2014-3555
Abstract 3562: Building workflows for gene fusion detection by RNA-seq
Authors: Gross Stephen , Watson Lisa , Pathak Smita , Khrebtukova Irina , Schroth Gary
Published: May 2015

Many cancers are associated with chromosomal rearrangements, which in some cases yield fusions between disparate genes and impact cancer phenotypes. Rapid discovery of gene fusions in cancer can facilitate a deeper understanding of specific cancers and may become an important diagnostic tool in clinical settings informing decisions on cancer treatment. An attractive and cost-effective method to identify and study gene fusions is by massively-parallel RNA sequencing (RNA-seq), which provides rich transcriptome profiling data and, with appropriate computational analysis, can discover fusions between transcribed genes. Herein we present data exploring the robustness of gene fusion detection by RNA-seq on Illumina sequencing platforms with TopHat-Fusion, a RNA-seq read aligner specifically designed to detect expressed gene fusions. Using a set of synthetic fusion transcripts to spike in to human RNA samples, we demonstrate quantitative detection of gene fusions by RNA-seq. Additionally, we report the sensitivity of gene fusion discovery across different RNA-seq library preparation methods, including mRNA enrichment by poly-A selection, rRNA-depletion, and exome enrichment by targeted capture. This data helps build workflow recommendations for the detection and study of gene fusions by RNA-seq.

Citation Format: Stephen Gross, Lisa Watson, Smita Pathak, Irina Khrebtukova, Gary Schroth. Building workflows for gene fusion detection by RNA-seq. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3562. doi:10.1158/1538-7445.AM2014-3562
Abstract 3563: New tools for RNA-sequencing of FFPE tumor samples
Authors: Watson Lisa , Pathak Smita , Gross Stephen , Khrebtukova Irina , Schroth Gary
Published: May 2015

Next generation RNA sequencing is an ideal platform to leverage archival formalin-fixed paraffin-embedded (FFPE) tissue samples for cancer research, as it offers extensive functional genomic information including transcriptome expression profiling, differential expression analysis and fusion detection. However, both RNA degradation from the formalin fixation process as well as limited sample material, pose major challenges for RNA sequencing of FFPE samples. Here we describe a novel method for generating whole-transcriptome RNA sequencing libraries from moderate to poor quality FFPE samples starting with as low as 20ng total RNA. This approach provides increased transcriptome coverage and robust measurement of gene expression levels compared to currently available FFPE RNA sequencing methods. Furthermore, we demonstrate that we can detect both known and novel gene fusions in clinical tumor samples that are potential drivers of disease. We anticipate this new technology will enable functional genomic studies on numerous FFPE tumor samples that would not previously be possible, providing invaluable information for developing cancer diagnostics and therapeutics.

Citation Format: Lisa Watson, Smita Pathak, Stephen Gross, Irina Khrebtukova, Gary Schroth. New tools for RNA-sequencing of FFPE tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2014-3563
Abstract 3569: Transcriptional signatures in human melanoma epitope MART-127-35 specific TCR-engineered T cells
Authors: Singh Prashant , Chakraborty Nitya G. , Farooq Umar , Hegde Upendra P. , Everson Richard , Dorsky David I. , Mukherji Bijay
Published: May 2015

Adoptive Cell Therapies (ACT) with T cell receptor-engineered (TCReng) T cells have shown promising results. Unfortunately, for a fuller understanding of their biology, currently available immunological assays have proven inadequate. Given that RNA-Seq using Next Generation Sequencing (NGS) offers substantial opportunities, we carried out NGS of the MART-127-35 specific TCReng human primary CD8+ and CD4+ T-cells following cognate stimulation, to obtain a comprehensive understanding of the transcriptional signatures underlying their biology.

Data analyses revealed approximately 30,000 transcripts, a sizeable number of which showing up- or down-regulation covering virtually all relevant aspects of T cell biology (i.e., effector function, differentiation, proliferative activities, apoptosis, etc.) when stimulated by the cognate epitope. The transcriptional profiles of the TCReng CD8+ and CD4+ T cells were found to be remarkably similar. Additionally, 72,000 known isoforms and about 20,000 previously undescribed isoforms were detected. Interestingly, a number of long noncoding RNAs (lncRNA) - some with known functions (e.g., NEAT1 and MALAT1, both implicated in RNA editing, were up-regulated in the two cell types), and others with unknown function (e.g., GAS5, MIAT, and FTX showing modulations in expression), were also detected in both cell types. Although a comprehensive knowledge of the transcriptional signatures and a clear understanding of the spliced variants, the novel isoforms, and the lncRNAs in T cell biology will require further work, our study shows that NGS of specific epitope reactive T cells will be a very useful tool in deciphering the transcriptional controls underlying the function and the fate of epitope specific anti-tumor T cells.

Citation Format: Prashant Singh, Nitya G. Chakraborty, Umar Farooq, Upendra P. Hegde, Richard Everson, David I. Dorsky, Bijay Mukherji. Transcriptional signatures in human melanoma epitope MART-127-35 specific TCR-engineered T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3569. doi:10.1158/1538-7445.AM2014-3569
Abstract 4447: Generation of a conditional S194A mutant RalA allele in mice
Authors: Crowe Matthew S. , Counter Christopher M.
Published: May 2015

The Ras family of small-GTPases represent the most frequently mutated oncogene in human cancer. Oncogenic Ras activates RalGEFs, which converts the Ras like small GTPases RalA and RalB to their active GTP-bound state to promote tumorigenesis. Despite being highly related, RalA generally tends to be more important in tumor growth while RalB has been implicated in metastasis. In regard to a mechanism underlying these functional differences, we note that RalA, but not RalB, is phosphorylated by the mitotic kinase AuroraA at Serine 194, and this phosphorylation is necessary for Ras-driven tumorigenesis. This phosphorylation has been found to result in an accumulation of RalA in mitochondrial fractions and an increase in mitochondrial fission and segregation during cytokinesis. Despite these encouraging observations, the role of this seemingly critical phosphorylation event in an actual in vivo setting was completely unknown. Thus, to determine the physiological consequence of this phosphorylation in vivo, we generated a mouse with a Cre-inducible S194A conditional Rala allele. We now report that these mice express wild-type RalA in the absence of Cre, but upon Cre-mediated recombination, convert wild-type exon 5 of Rala to one encoding the S194A mutant version. We propose to next investigate the consequences of blocking this critical posttranslational modification on normal development and on oncogenic Kras-driven tumorigenesis.

Citation Format: Matthew S. Crowe, Christopher M. Counter. Generation of a conditional S194A mutant RalA allele in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4447. doi:10.1158/1538-7445.AM2014-4447