bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2953: Involvement of HDAC11 in cell division cycle
Authors: Bagui Tapan K. , Sharma Savitha , Pledger W. J.
Published: June 2012

Histone deacetylases (HDACs) are a group of enzymes that catalyze removal of acetyl groups from a ε-N-acetyl lysine amino acid on histones. A number of non-histone proteins are also known to be deacetylated by these enzymes. Hence, these proteins are now also being referred to as lysine deacetylases (KDAC).

Based on function and DNA sequence similarity, HDAC proteins are classified in four groups; Class I, Class II, Class III and Class IV HDACs. HDAC11, the only member of Class IV HDAC, shows a DNA sequence similarity with both Class I and Class II HDACs.

In general, HDAC proteins are known as transcriptional inhibitors and are involved in cellular growth, development and apoptosis. Unlike other HDACs, function of HDAC11 is not widely studied. Although this enzyme is reported as a transcriptional inhibitor, little is known about its role in connection to cell division cycle or cell density. Analysis of our microarray data shows that Balb/C 3T3 cells, stimulated with PDGF and plasma, contain considerably reduced level of HDAC11 transcript compared to that in the quiescent Balb/C 3T3 cells. The protein level under similar conditions mimics the pattern of transcript level. These results indicate that as cells leave quiescence state HDAC11 expression is down-regulated or may be reduction of HDAC11 expression causes quiescent cells to enter cell cycle. It may also be possible that cell-cell contact inhibition positively regulates HDAC11 expression and as the cell grows in presence of PDGF and plasma, the contact inhibitory property is lost which in turn causes the reduction of the protein expression. Further work is in progress to elucidate the role of HDAC11 with respect to cell cycle regulation or cell density.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2011-2953
Abstract 2972: In vitro quantification and modeling of DNA damage by histone H2A after exposure to gemcitabine and a Chk1 inhibitor
Authors: Kang Xiaolin , Kindt Erick , Yamazaki Shinji , Troche Gabriel E. , Spilker Mary E.
Published: June 2012

Background: Gemcitabine is a nucleoside analog used as an anti-cancer chemotherapeutic drug. To enhance its disruption of DNA replication and repair, Chk1 inhibitors are being developed for use in combination with Gemcitabine and other cytotoxic agents. Evaluation of these therapies with mechanistic biomarkers can be an important part of the drug development process. One such biomarker is the phosphorylation of histone H2A (γH2AX), which is a DNA damage response biomarker. An ELISA based assay has been developed to quantify γH2AX. This quantitative assay provides data that is incorporated into a mathematical model relating drug exposure to DNA damage and cell viability.

Experiment Design: MiaPaCa-2 cells, a human pancreatic tumor cell line, were subjected to Gemcitabine alone (0.1 to 5uM for 2 hours), a Chk1 inhibitor alone (0.1-5uM for 2 – 48 hours), or the Chk1 inhibitor in combination with 0.5 and 5uM Gemcitabine. Cellular concentrations of Gemcitabine and the Chk1 inhibitor were detected with LC/MS. DNA damage was assessed by the γH2AX ELISA at 2, 6, 24 and 48 hours after the start of incubation with drug. In addition to γH2AX, cell viability was measured by cell number at 48 hours. A mathematical model was developed to quantify the modulation of γH2AX when exposed to each drug separately and in combination.

Result: Transient exposure of the cells to Gemcitabine resulted in an increase in γH2AX at later times (24 and 48 hours). The Chk1 inhibitor resulted in a dose dependent increase in γH2AX, with a greater effect being observed at later times of exposure. The combination therapy showed a greater response than Gemcitabine or Chk1 alone. However it was observed that higher doses of Gemcitabine and Chk1 in combination did not always yield the largest γH2AX response or the greatest effect on cell viability. This may be due to cellular resistance and is still being investigated.

Conclusion: The ELISA based assay for γH2AX provides a quantitative assessment of the level of DNA damage. This information can then be utilized in developing mathematical models of drug action alone and in combination with other drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2972. doi:10.1158/1538-7445.AM2011-2972
Abstract 2982: Impact of the RB-pathway in breast cancer heterogeneity and therapeutic response
Authors: McClendon A. Kathleen , Ertel Adam , Dean Jeffry , Thangavel Chellappagounder , Knudsen Karen E. , Witkiewicz Agnes K. , Knudsen Erik S.
Published: June 2012

Molecular heterogeneity in breast cancer is associated with differential prognosis and response to therapy. However, in spite of extensive molecular classification, pathways that can be specifically targeted to understand the basis of disease and improve treatment remain poorly understood within specific subtypes of breast cancer. The retinoblastoma tumor suppressor (RB) pathway is deregulated in most cancers; however varied mechanisms of inactivation have significant impacts on tumor biology. Here novel mechanistic and preclinical models were combined with analyses of tumor specimens to decipher the differential impact of the RB-pathway within breast cancer subtypes and as a specific target for patient stratification and therapeutic targeting.

Initial gene expression datasets encompassing over 2000 breast cancer patient samples were used to demonstrate a striking dichotomy in the mechanisms of RB functional inactivation within subtypes of disease. In ER-positive breast cancer, disruption of RB-pathway function was associated with poor prognosis and failure of endocrine therapy. Functional studies demonstrate that such therapeutic failure largely occurs in the presence of an intact Rb gene, as a result of deregulated mitogenic signaling. In such models of hormone refractory disease we demonstrate that pharmacological activation of RB is a potent second-line therapy. In contrast, in ER-negative tumor populations RB inactivation occurs in 40-50% of cases. This event yields complete loss of RB protein, as occurs through genetic and epigenetic mechanisms. Strikingly, in ER-negative disease RB loss is associated with improved response to chemotherapy and longer relapse-free survival. The basis for improved response involves both deregulation of cell cycle checkpoints and apoptotic processes. Ongoing analyses are using synthetic-lethal and combination-therapy approaches to rationally target RB-status in the management of breast cancer.

In total, these studies define a clear role for RB-pathway as a determinant of tumor heterogeneity in breast cancer that can be leveraged to more effectively treat breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2982. doi:10.1158/1538-7445.AM2011-2982
Abstract 2987: A critical role of translocated promoter region (Tpr) in chromosome segregation and cell division in cancer
Authors: Funasaka Tatsuyoshi , Nakano Hiroshi , Wong Richard
Published: June 2012

Cancer cells accumulate the many genetic abnormalities required for cancer development. Abnormal chromosome is often observed in cancer cells and is thought to be due to chromosome segregation errors during mitosis. In eukaryotic cells, the faithful segregation of daughter chromosomes during cell division depends on formation of a microtubule-based bipolar spindle apparatus. Nuclear pore complexes are massive multiprotein channels responsible for traffic between the nucleus and cytoplasm, and are composed of approximately 30 proteins termed nucleoporins. Several components of the nuclear pore complex/nucleoporins play critical roles in orchestrating the rapid remodeling events that occur during mitosis. Our recent studies revealed that the nucleoporin, Rae1, plays critical roles in maintaining spindle bipolarity. Here, we show association of another nucleoporin, termed Tpr (translocated promoter region), with the molecular motors dynein and dynactin, which both orchestrate with the spindle checkpoints Mad1 and Mad2 during cell division. Overexpression of Tpr enhanced multinucleated cell formation. RNA interference-mediated knockdown of Tpr caused a severe lagging chromosome phenotype and disrupted spindle checkpoint proteins expression and localization. Next, we performed a series of rescue and dominant negative experiments to confirm that Tpr orchestrates proper chromosome segregation through interaction with dynein light chain. Our data indicate that Tpr functions as a spatial and temporal regulator of spindle checkpoints, ensuring the efficient recruitment of checkpoint proteins to the molecular motor dynein to promote proper anaphase formation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2987. doi:10.1158/1538-7445.AM2011-2987
Abstract 2988: Regulation of mitosis and taxane response by Daxx
Authors: Giovinazzi Serena , Lindsay Cory , Morozov Viacheslav , Han Hyo , Ishov Alexander
Published: June 2012

Current theories suggest that mitotic proteins are essential for cellular response to taxanes (paclitaxel and docetaxel), a widely-used family of chemotherapeutic compounds. We found that absence or depletion of protein Daxx increases cellular resistance to paclitaxel _a common trait of patients diagnosed with breast cancer and other malignancies. Particularly, we observed that 1) paclitaxel induced cell death, in both human breast cancer and mouse cell lines, depends on the level of Daxx; 2) cells become paclitaxel-resistant upon experimental depletion of Daxx; 3) breast cancer specimens accumulate differential level of Daxx; 4) docetaxel response in breast cancer patients correlates with level of Daxx mRNA. We also found that Daxx is important for the proper progression of mitosis in human cells: duration of mitotic stages is altered in cells stably depleted of Daxx which also show increased stability of cyclin B1. Thus, protein Daxx, that was previously characterized as a transcription repressor and an apoptotic-related protein, may also have a novel function in mitosis progression as a prometaphase checkpoint release protein in control conditions and upon mitotic stress.

In order to understand the mechanism by which Daxx orchestrates mitosis progression and identify new potential targets responsible for taxane resistance, we adopted a functional proteomic approach to identify Daxx-associated mitotic complex. We optimized the protocol for Tag based affinity purification in a way to obtain Daxx expressed downstream of FLAG Tag in tandem with HA Tag (FH) and a Thrombin Cleavage Site (TCS). HEp2 cells transduced with pOZ-FH-TCS-Daxx and the empty pOZ-FH plasmid (control), synchronized by double thymidine block and next arrested in pro-metaphase with paclitaxel, were used to purify Daxx containing mitotic complex. Mitotic specific Daxx interacting proteins were identified by SDS-PAGE followed by mass spectrometry.

The functional characterization of newly identified Daxx mitotic partners will shed lights on the complex mechanism/phenomena of taxanes resistance and ultimately offer the molecular basis for successful treatment strategies of breast cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2011-2988
Abstract 2992: Mitotic role for Pten in accurate chromosome segregation
Authors: van Ree Janine H. , Jeganathan Karthik B. , Jin Fang , van Deursen Jan M.
Published: June 2012

During mitosis, sister chromatids are separated and equally divided over two daughter cells. If missegregation of whole chromosomes occurs, progeny cells with an abnormal number of chromosomes will arise. This is referred to as aneuploidy, a hallmark of most human cancers.

We found that inactivation of a single Pten allele in mouse splenocytes and mouse embryonic fibroblasts (MEFs) is sufficient to cause aneuploidy, suggesting a role for Pten in the chromosome segregation process. Upon further study by live cell imaging of haploinsufficient and null Pten MEFs, we indeed found many missegregation errors (59% and 79% respectively, compared to 15% in wildtype MEFs). These abnormalities were independent from the AKT pathway since overexpression of a myristoylated, constitutively active form of AKT in wildtype MEFs resembled a wildtype instead of a Pten heterozygous phenotype. In addition, overexpression of a catalytic mutant Pten (C124S) in Pten+/- MEFs corrected the missegregation errors. We therefore propose a novel tumor-suppressive function of Pten to prevent whole chromosome instability, independent of the increased cell proliferation and survival caused by the activation of AKT.

BubR1 is a regulator of kinetochore-microtubule attachment and a potent inhibitor of Cdc20, a key co-activator of the anaphase promoting complex/cyclosome (APC/C) that drives cells into anaphase by targeting the anaphase inhibitors cyclin B1 and securin for degradation by the 26S proteosome. We found that overexpression of BubR1 completely restores accurate chromosome segregation in Pten+/- MEFs, alluding to a specific role for Pten in mitosis. As a first step to test the hypothesis that Pten's mitotic role in preventing aneuploidization represents a key tumor suppressive function of this phosphatase, we bred a Flag-tagged BubR1 transgene into Pten+/- mice. We are currently in the process of studying the impact of the transgene on the development of prostatic intraepithelial neoplasia (PIN). Consistent with our hypothesis, preliminary analysis of two mice shows that BubR1 overexpression considerably decreases PIN lesion multiplicity. Data from the complete analysis of our double transgenic mouse cohort will be presented at the meeting.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2011-2992
Abstract 2999: Role of heterogeneous nuclear ribonucleoprotein A/Bs in telomere maintenance
Authors: Wang Tzu-Chien V. , Huang Pei-Rong , Yeh Yuan-Ming , Hung Shu-Chen
Published: June 2012

Heterogeneous nuclear ribonucleoproteins (hnRNPs) constitute a large family of proteins that associate with nascent pre-mRNAs, packaging them into hnRNP particles. In addition to their action as RNA-binding proteins, several members of the hnRNP family are thought to play roles in telomere maintenance because they are known to bind to telomeric DNA and to TERRA RNA, which is now identified as an integral component of telomeric heterochromatin. Using affinity purification and proteomic approach, we have previously shown that the members of hnRNP A/B family are the most abundant proteins in the nuclear extracts that bound specifically to G-strand telomeric DNA. To address if members of hnRNP A/B family are essential components of a functional telomere (i.e., does hnRNP A/Bs play a structural role) and to determine the functional overlap among members of hnRNP A/Bs in telomere length regulation, we initiated to investigate the biological effects of single, double, or triple knock-down of hnRNP A1, A2/B1 or A3. Preliminary results obtained from transient depletion of single hnRNP A1, A2 or A3 by RNA interference (RNAi) indicated that depletion of hnRNP A2 or A3 significantly reduced cell growth and colony formation. The percentage of cells in the sub-G1 phase was increased following the knockdown of hnRNP A2 or A3. In addition, the expression of S-phase kinase-associated protein-2 (skp2) was decreased in the transient knock-down cells. Surprisingly, however, studies of stable clones derived from single knock-down experiments indicated that knock-down of hnRNP A2, but not A1 or A3, produced a profound effect on cellular growth. This effect does not appear to result from reduced viability and/or apoptosis. We noted that an increased expression of hnRNP A1 was always observed in the stable clones depleted of hnRNP A3. Similarly, increased expression of A3 was observed in the cells depleted of hnRNP A1, suggesting that hnRNP A1 and A3 may have functional redundancy in vivo. Works are in progress to determine whether the anti-proliferation effects of depleting hnRNP A/B may result from telomere dysfunction, and to investigate the functional overlap between hnRNP A/Bs using double knock-down approach.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2999. doi:10.1158/1538-7445.AM2011-2999
Abstract 3013: Study of three-dimensional cell culturing influence on DNA methylation profile
Authors: López-Serra Paula , Martín-Subero Ignacio , Esteller Manel
Published: June 2012

In this study, we purpose the analysis of the DNA methylation profile of four different human breast cell lines, two non transformed and two tumoral, taking into account the influence of the environment in cell culturing.

The extracellular matrix was initially thought to serve as nothing more than a physical support for the cells. However, nowadays is very well known that the extracellular matrix is a key component in cell biology, regulating cell biology and tissue homeostasis. This suggests that there is a bidirectional crosstalk between the cells and the surrounding extracellular matrix.

Cell culturing is a commonly used technique in all areas of biology research. Usually, adherent cells are spreading in two-dimensional plates where they growth attached to the plastic surface in two directions. Mammary epithelial cells that are cultured on this conventional way fail to form acinar-like structures and loss tissue specific protein expression. Many of the microenvironment influences can be restored in cell culturing models by using three-dimensional cultures in extracellular matrix gels. 3D culturing of epithelial cells can help to reconstruct the acinar morphology seen in vivo and restore some mammary-specific function, like milk proteins production.

DNA methylation plays a significant role in the epigenetic regulation of chromatin structure and gene expression. In this work, we tried to analyze the DNA methylation profile of four different human breast cell lines growth in 2 and 3 dimensions to assess the influence of DNA methylation in the two models. HMEC (finite lifespan human mammary epithelial cell line), MCF10A (spontaneously immortalized, but not transform, human breast epithelial cell line), MCF7 (human breast adenocarcinoma cell line, metastasic) and MDA-MB-231 (human breast adenocarcinoma cell line, metastasic) were growth in plates with and without MatrigelTM basement Membrane Matrix Growth Factor Reduced (BD). 3D culturing was done by spreading the cells in the matrix surface and allowing them to form the characteristic acini of the mammary gland. Morphological differences are evident between the 2 and 3D culture models, being much more accurate the 3D model from a phenotypic point of view. DNA mehtylation status was done by illumina's Infinium Microarrays, comparing the methylation profile of each cell line growth in 2 and 3D. We determined in this way which genes are differentially methylated in 2 and 3 dimensions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3013. doi:10.1158/1538-7445.AM2011-3013
Abstract 3014: Quantitative accuracy of Illumina HumanMethylation27 Infinium BeadChip data assessed by pyrosequencing
Authors: Huang Zhiqing , Yamaguchi Ken , Berchuck Andrew , Murphy Susan K.
Published: June 2012

Purpose. Aberrant DNA methylation can lead to changes in gene expression, and this alteration is highly relevant in the context of cancer. The ability to accurately assess DNA methylation status is therefore of great importance in health and disease. The Illumina HumanMethylation27 Infinium BeadChip allows for analysis of whole genome DNA methylation by interrogating the methylation status of 27,578 CpG sites from the promoter regions of 14,475 annotated genes. However, one potential limitation of this platform as specified by the manufacturer is the inability to detect a β value difference between specimens of <0.2 (∼20% methylation). The present study assessed the quantitative nature of the Infinium platform through independent validation using a pyrosequencing approach.

Procedures. Genomic DNA from 46 ovarian cell lines and 4 cultured normal cells, including ovarian surface epithelium and endometriotic cells, was modified with sodium bisulfite using the EZ DNA methylation kit from Zymo Research. Bisulfite conversion efficiency was confirmed by pyrosequencing at two chromosomal loci. Comparisons between normal and cancer cells identified differentially methylated genes using criteria of p0.3. Ten candidate differentially methylated genes (C14orf105, HNF1 β, HNF1α, KIF12, MIA2, PAX8, SERPINA6, SGK2, SRC, and TM4SF4) were selected for validation. Pyrosequencing assays were designed to allow for direct comparison of the same CpG site(s). The relationship between pyrosequencing and Infinium data was evaluated using correlation analyses.

Results. Bisulfite conversion was >97% at BRCA1 (chr17) and IGF2 (chr11). Hypermethylation of 1,162 genes (1,452 CpG sites) and hypomethylation of 63 genes (74 CpG sites) was found in ovarian cancer cell lines as compared with cultured normal cells. The β values for 10 genes from Infinium analysis and percent methylation by pyrosequencing of the same CpG site(s) were highly correlated (R>0.98, p0.91, p<0.001). There was a significant positive correlation between platforms when data for all ten genes were grouped into 0-20%, 21-40%, 41-60% 61-80% and 81-100% bins (R value range, 0.32-0.73; p< 0.00002).

Conclusions. Ovarian cancer cell lines exhibit significantly increased methylation as compared with normal cultured cells using the HumanMethylation27 Infinium BeadChip assay. The strong correlations between the Infinium and pyrosequencing data at the same CpG sites suggest that the Infinium platform is quantitative, lending support for use of this technology for assessment of methylation differences among specimens analyzed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3014. doi:10.1158/1538-7445.AM2011-3014
Abstract 3015: DNA methylation biomarkers for lung cancer diagnostics: Validation in bronchial washings
Authors: Liloglou Triantafillos , Nikolaidis George , Raji Olaide , Field John Kirpatrick
Published: June 2012

Epigenetic reprogramming is a characteristic of virtually of lung tumors. DNA methylation is unique for biomarker utilization in body fluids due to its chemical stability and the high frequency of abnormal hypermethylation in lung tumors. Despite the evidence from a wide range of studies in this field, very little is progressing to clinical exploitation, mainly due to the insufficient preclinical and clinical validation. In this study we have undertaken extensive preclinical validation of DNA methylation biomarkers in bronchial washings from 320 lung cancer cases and 320 age/sex matched controls organized in training (n=150 in each arm) and validation (n=170 in each arm) sets. Quantitative methylation specific PCR (qMSP) assays were developed and optimized through multiple sets of methylation standards. The hypermethylation detection threshold was set to 0.5% based on the reproducibility of signal detection.

Among the promoters tested p16, RASSF1, WT1, RARbeta, TERT and CYGB proved to be highly discriminating between cases and controls while CDH13, TMEFF2, p73 and DAPK did not demonstrate a significant difference. Using a criterion of at least two positives from the discriminating markers in the validation set, this panel of six markers demonstrated an overall 70.5% sensitivity and 97.7% specificity. It is of note that when combining methylation biomarker with cytology, the positive detection rate was 81.5%. Regarding tumor histology, the detection sensitivity (combining training and validation sets) was higher in small cell carcinomas (95.1%) and squamous carcinomas (65.2%) than in adenocarcinomas (51.6%).

Our results clearly demonstrate the clinical potential of DNA methylation in lung cancer diagnostics, although it is profound that this panel must be enriched by additional promoters. It also becomes apparent that combined multi-center studies of increased statistical power must be undertaken in order to promptly validate biomarker panels for clinical implementation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2011-3015
Abstract 304: Predictive factors of pathological complete response by neoadjuvant chemotherapy for operable breast cancer
Authors: Ishikawa Takashi , Shimizu Daisuke , Sasaki Takashi , Morita Satoshi , Tanabe Mikiko , Ota Ikuko , Kawachi Kae , Chishima Takashi , Ichikawa Yasushi , Endo Itaru
Published: June 2012

Background: Nonanthracycline regimens with taxanes have become to be used more frequently than before. Thus, it has become important to consider sensitivities to these two drugs in individual patients.

Aim: To examine the association between the pathological response of each regimen and several pathological factors and whether individualized treatment based on HER2 may improve the overall pathological effects.

Patients and methods: 1) Retrospective study: 170 patients treated with standard regimens consisted of anthracycline and/or taxan prior to surgery. 2) Prospective study: 18 patients with HER2 overexpression received 4 cycles of anthracycline and cyclophosphamide (AC), while 45 pathients without HER2 expression received 4 cycles of docetaxel and cyclophosphamide (TC). For these 2 studies, the overall pCR rates were examined. The association of pCR with pathological factors including estrogen- and progesterone-receptor (ER and PgR), HER2, nuclear grade, Ki-67, P-53 and Topoisomerase IIα (TopoIIα) etc. were also examined.

Results: 1) The overall pCR rate was 24.2 % (41/170). Multivariate analysis with factors revealed that ER and nuclear grade were significantly associated with pCR. 2) The overall pCR rate was 36.5% (23/63), with pCR rates of 27.8% (5/18) and 40.0% (18/45) for the AC and TC regimens, respectively. TC regimen induced a pCR in most triple-negative cancer cases (15/19). Only low expression of ER maintained statistical significance as a predictor of pCR for the TC regimen.

Conclusions: Individualized HER2-based treatment improved the efficacy of neoadjuvant chemotherapy, probably because HER2 is, at least at present, the most promising predictor for the efficacy of anthracyclines. Further studies with predictive factors including TopoIIα by FISH are now under investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 304. doi:10.1158/1538-7445.AM2011-304
Abstract 3053: Receptor tyrosine kinase AXL as a new target for prostate cancer therapy
Authors: Paccez Juliano D. , Vasquez Gabriela , Tamura Rodrigo E. , Duncan Kristal , Vasconcellos Jaira , Wiesner Theresa , Libermann Towia , Zerbini Luiz F.
Published: June 2012

Axl is a receptor tyrosine kinase of the family of TAM receptors (which includes TYRO3 and MER) and plays roles in different types of cancer. It is highly expressed in metastatic colon carcinoma, gastric, uterine endometrial and certain types of breast cancer, and sarcoma. As Axl is upregulated in several metastatic cell types it may play a role during invasion and metastasis. Tyrosine kinases (TKs) represent a major class of proto-oncogenes and are involved in tumor growth as well as progression and metastasis of cancer cells. TKs are being actively studied as targets for therapeutic intervention and several of this tyrosine kinase have shown efficacy in clinical trials. Prostate cancer has become the most common solid cancer in older men and is one of the most frequent causes of cancer deaths. Although androgen ablation therapy, surgery and radiation therapy are effective for the treatment of local prostate cancer, there is no effective treatment available for patients with the metastatic androgen-independent disease.

In this work we demonstrated the role of Axl in prostate cancer progression. Using real time PCR to access the level of several tyrosine kinase receptors expression in prostate cancer cell and human tissue, we have identified the tyrosine kinase receptor Axl as a potential target for prostate cancer therapy. Axl has consistent overexpression across prostate cancer cell lines and human prostate tumor tissue providing a model for testing the targeting of Axl. Preliminary data shows a significant increase of Axl expression was found in metastatic PCa cells and clinical samples (48% of adenocarcinomas of prostate when compared with normal prostate tissue). Interestingly, microarray analysis in addition to pathway analysis of PCa cells infected with lentivirus encoding siRNA against Axl revealed that some survival pathways are inhibited, but strikingly all members of the NF-κB pathway are down regulated. Furthermore, the blockage of Axl gene expression strongly inhibits proliferation, migration and invasion of PCa cells. Pilot studies in animal models demonstrate that inhibition of Axl reduces dramatically tumor formation. since our xenograft subcutaneous model in MF5 nude mice demonstrate that blockage of Axl reduces tumor formation by 49.5%.

Taken together these data demonstrated that Axl plays role for migration, invasion and tumor development and can be used as marker for invasive and metastatic tumors. Our data also indicates that Axl can be used as a target for drug therapies since its inhibition leads to induction of apoptosis and blocks cell proliferation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2011-3053
Abstract 3059: CCL2 overexpression in an epithelial ovarian cancer cell line results in reduced tumorigenic potential
Authors: Wojnarowicz Paulina M. , Gambaro Karen , Desaulniers Kim Leclerc , de Ladurantaye Manon , Provencher Diane , Mes-Masson Anne-Marie , Tonin Patricia N.
Published: June 2012

Chromosome 17 abnormalities are frequently observed in epithelial ovarian cancer (EOC), suggesting that one or more genes mapping to this chromosome contribute to EOC development and/or progression. We have previously performed a comparative transcriptome analysis of primary cultures of normal ovarian surface epithelial (NOSE) cells and malignant, serous subtype ovarian tumor (TOV) samples in order to identify differentially expressed chromosome 17 genes. CCL2, at 17q12, was significantly differentially underexpressed at least 3-fold in all TOV samples. We have confirmed underexpression in an independent series of 99 serous EOC tumor samples. Interestingly, CCL2 was identified as a significantly up-regulated gene occurring as a consequence of tumor suppression in the characterization of a novel EOC cell line model developed by transferring chromosome 3 fragments into the tumorigenic OV-90 EOC cell line. We have further characterized CCL2 expression in a series of EOC cell lines that differ in their tumorigenic potential, and have shown the lowest levels of gene expression occur in tumorigenic cell lines. DNA sequencing did not reveal any evidence of mutations. To determine if overexpression affects tumorigenicity or growth characteristics, we have generated stable overexpressing OV-90 clones using a commercially available pDream: CCL2 expression vector. Overexpression was verified by ELISA. In cell culture, the CCL2 overexpressing clones appeared less tightly packed and more elongated as compared with OV-90 cells. The cell viability of the CCL2 clones appears to be reduced when compared to the parental OV-90 cell line in XTT cell proliferation assays. The ability of the CCL2 clones to form colonies was also reduced as compared to OV-90 in a colony formation assay. There is also evidence of an increase in the percentage of senescent cells in the CCL2 clones based on a beta-galactosidase staining assay. Both CCL2 overexpressing clones exhibit latency in tumor formation in intraperitoneal tumorigenicity assays involving SCID mice as compared with the parental OV-90 cell line. This is the first report of stable CCL2 transfection into an EOC cell line. Our results suggest that CCL2 expression may reduce the tumorigenic potential of EOC cells, and draws attention to the role of the innate immune system and the tumor microenvironment in EOC development and progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3059. doi:10.1158/1538-7445.AM2011-3059
Abstract 306: Development of EC-DG as a molecular theranostic personalized medicine
Authors: Yang David J. , Ford Richard J. , Mendez Richard , Zhang Yin-Han , Bryant Jerry , Oh Chang-Sok , Huang Jack , Pham Lan , Kohanim Saady , Kim Edmund E.
Published: June 2012

Purpose: D-glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. We have then synthesized Tc-99m- ethylenedicysteine-glucosamine (EC-DG). We found Tc-99m-EC-DG was involved in cell proliferation in lung, breast and head and neck cultures and could assess breast cancer treatment outcome in vivo by planar scintigraphy. Tc-99m-EC-DG is a safe imaging agent in lung cancer patients. This study was amied to (1) assess a novel response to unlabeled rhenium-EC-DG (Re-EC-DG) involving the translation regulation of hypoxia inducible factor (HIF)-1alpha expression in lymphoma cells, and, (2) evaluate feasibility of using EC-DG for theranostic approaches in cancers.

Methods: For theranostic assessment studies, we synthesized cold Re-EC-DG. Re-EC-DG was synthesized via a two-step synthesis. The first step was to synthesize Re-EC by reacting rheniumoxo trichloride with EC. The second step was to react Re-EC with D-glucosamine tetraacetate, followed by de-acetylation. Twelve types of DLBCL cells were incubated with Re-EC-DG at various concentrations (0-10 mM) and TUNEL assays were used to determine cell apoptosis. To ascertain the mechanism of the anticancer properties for Re-EC-DG, DLBCL-LY10 cells were treated with Re-EC-DG (0-5 mM) for 48 hrs. Immunoblotting were then performed on nuclear extracts with 50 µg. For radiotheranostic assessment studies, 13762 breast tumor-bearing rats were imaged with In-111-EC-DG and tumor/muscle ratios were determined at 0.5-24 hrs. Radiation absorbed dose was estimated for the use of Y-90-EC-DG.

Results: There was a dose response relationship of Re-EC-DG inhibition in DLBCL cells. Extensive apoptosis was observed at 24 hrs in lymphoma cell cultures. Re-EC-DG showed significant tumorcidal activity compared to normal B-lymphocyte activity at doses >0.17 µmol. Re-EC-DG caused a decreased expression of HIF-1alpha under normoxic conditions in DLBCL-LY10 cells. Tumor-to-muscle ratios for In-111-EC-DG were 5.43±0.45 to 7.80±0.05 whereas In-111-EC had 3.24±0.32 to 4.64±0.16 at 0.5-24 hrs. Radiation exposure of In-111-EC-DG to whole body, blood-forming organs, gonads, and effective dose equivalent for a single dose at 5 mCi was below the limits of 3 rad annually and 5 rad total. The absorbed dose in all other organs was below the limits of 5 rad annually and 15 rad total.

Conclusion: EC-DG is a useful molecular theranostic compound. In-111-EC-DG has favorable dosimetry, providing a potential use of Y-90-EC-DG to treat cancers. Re-EC-DG inhibits HIF-1alpha expression and is an attractive anti-proliferation compound.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 306. doi:10.1158/1538-7445.AM2011-306
Abstract 3060: Target genes of chromosomal 9p13.3 amplification in prostate cancer
Authors: Latonen Leena , Leinonen Katri A. , Saramäki Outi R. , Visakorpi Tapio
Published: June 2012

The development and progression of cancer is caused by alterations that accumulate in the genome of malignant cells. We are studying genetic alterations occurring in prostate cancer, the most common male malignancy in Western countries. We have previously identified a frequent (39%) gain at 9p13-q21 in 13 prostate cancer xenografts and 5 cell lines by aCGH (Saramäki et al., Int J Cancer 119:1322-1329, 2006). Preliminary results indicate that in clinical prostatectomy samples, gains and amplifications are found in approximately 30% and 10% of the cases, respectively, and that the amplification frequency seems to increase with disease progression to hormone-refractory stage. The 9p13.3 amplification is also found from several breast cancer cell lines, raising the possibility that the amplification target gene(s) in this area may take part in breast cancer formation in addition to prostate cancer.

We aim at identifying the target gene of the 9p13.3 amplification in prostate cancer. A minimal region for the 9p13.3 amplification, determined by fluorescence in situ hybridisation (FISH), spans nearly 3.5 Mb and contains more than 40 known and hypothetical protein-coding genes. We initially screened mRNA expression levels by quantitative RT-PCR and chromosomal copy number alterations by FISH for these genes in 7 prostate cancer cell lines and 19 prostate cancer xenografts. Thus, we narrowed down the list of amplification target candidates to eight protein-coding genes (C9orf25, GALT, PIGO, UBAP1, UBAP2, UBE2R2, UNC13B and VCP). We have performed siRNA-mediated downregulation of these genes in cell lines with either a normal copy number status or a gain/amplification in 9p13.3 (prostate cancer cell lines PC-3 and 22Rv1; breast cancer cell lines MCF-7 and BT-474). Downregulation of several of these genes decrease the proliferation rate of the cells significantly (GALT, PIGO, UBAP1, UBAP2, VCP). As decreased levels of VCP induce cell death in PC-3 cells, downregulation of UBAP1 induces a cell cycle arrest in the G2/M phase. In addition, invasion of PC-3 cells in Boyden chamber assay is affected by knock-down of GALT and PIGO. Genes exhibiting alterations in the siRNA assays have been further analyzed for their expression in clinical prostate cancer tumor material by qRT-PCR and immunohistochemistry. These genes are also studied by overexpressing their cDNAs in cell lines with normal 9p13.3 copy number status and screening for altered cellular growth, survival and invasion in vitro. We aim at identifying a novel diagnostic and/or prognostic marker for prostate cancer, and potentially also a target for new cancer therapies, from the 9p13.3 region.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3060. doi:10.1158/1538-7445.AM2011-3060
Abstract 3064: A novel method for mutation analyses using genomic DNA obtained from IHC-stained sections
Authors: Desai Rupal M. , Patel Rajesh , Amler Lukas , Stern Howard , Raja Rajiv
Published: June 2012

Biopsies obtained from patients are often limited in size. The ability to perform multiple diagnostic assays is dependent on the quantity and quality of tissue samples available from patients. Over the last few years, our laboratory has focused our efforts on developing novel methodologies for increased efficiency in use of tissue samples. Here we describe a method for performing immunohistochemistry (IHC) and genotyping using the same tissue section. We isolated genomic DNA (gDNA) from IHC-stained and unstained formalin-fixed paraffin-embedded (FFPE) tissue sections obtained from the same paraffin block. Quality and quantitylity of gDNA obtained from either sections were compared. We demonstrate that quality of gDNA obtained is acceptable for down-stream analysis using PCR techniques. We also show comparable specificity and sensitivity in detecting mutations using either sources of gDNA. This method has proven highly valuable in performing mutation analysis in certain tissue types such as lung cancer where tissue can be extremely limited.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2011-3064
Abstract 3066: Direct detection and enumeration of circulating tumor cells (CTCs) in blood by CastRT-qPCR
Authors: Deng David X. , Sprout Scott , Bao Yun , Merrill David , Brzoska Pius , Wang Yu , Chen Caifu
Published: June 2012

Statistical data suggest that only approximately 25% of cancer patients will respond to the same treatment. Enumeration and molecular characterization of circulating tumor cells (CTCs) promise to be valuable for cancer cell diagnosis, survival prognosis, and treatment guidance. However, CTCs are very rare. There is no sensitive and specific assay available for analyzing CTCs from whole blood without extensive enrichment processes. Sample enrichment causes target cells loss, expensive and time-consuming. Here we report a highly specific competitive allele-specific TaqMan-based RT-qPCR (castRT-qPCR) for rare copies of DNA mutation detection. CastPCR can detect rare copies of mutant alleles for up to one in 10 million with a wide dynamic range of more than 6-log orders and <5 copy sensitivity. We have successfully designed and validated cast-PCR assays for more than 80 cancer-related SNPs of KRAS, EGFR, BRAF, KIT, pTEN, p53 and other oncogenes or tumor suppressor genes. CastRT-PCR was used to specifically detect and enumerate CTCs in blood samples with lung cancer cell lines spiking-in and lung cancer patient blood without prior biochemical enrichment processes. EGFR mutation (p.L858R) was detected in all 5 blood samples from late stage lung cancers. Approximately 50% of circulating lung tumor cells with positive EGFR p.L858R mutation had positive EGFR p.T790M mutation. In normal blood samples spiked in cell lines of known Kras and EGFR mutation at the level of 10 cells/mL of whole blood (close to actual patient samples), CastRT-PCR accurately detected the known mutations. Our data suggest that digital castRT-qPCR can be used to enumerate CTCs and detect cancer-related mutations in the blood, which paves the way for future individualized cancer treatment by monitoring CTC level and molecular characterization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2011-3066
Abstract 3070: Simultaneous amplification of multiple genetic/epigenetic tumor markers in a single reaction for high throughput genotyping and aberrant methylation profiling
Authors: Liu Liansen , Liu Chunmei , Stevens Junko , Wang Jonathan T.
Published: June 2012

Matching appropriate therapeutic regimes with tumor genotypes and epigenotypes is of great medical and economical importance. A prerequisite for this approach is thorough and accurate genotype and epigenotype profiling of tumor specimens, which should be performed in precisely defined homogeneous tumor cell populations with minimum non-tumor cell contaminations. However, this can be very challenging due to the genetic and epigenetic heterogeneities and the scarcity of homogeneous tumor cells within a tumor specimen. Multiplex PCR allows high throughput profiling by maximizing the number of targets/reaction in each sample and can reduce the limitations of such sample shortage. Although several multiplex PCR kits have been commercially available for this purpose for some time, their usefulness is restricted by small amplicon size range, less satisfactory amplification uniformity of different amplicons, and occasional failure in amplifying some amplicons, or false positive amplifications. To solve these problems, we have devised a highly specific, optimization-free multiplex PCR formulation (Applied Biosystems Platinum® Multiplex PCR Master Mix) with novel reaction chemistry. We show here that this new formulation is excellent for simultaneous amplification and high throughput profiling of SNPs, deletions, insertions and their wild types of many targets in a single reaction in terms of robustness, uniformity, amplicon size range, compatibility with amplicon GC contents, and the reduction of non-specific primer dimers and false positive/negative products in multiplex PCR. In combination with a primer system of varied lengths and compositions of 5’ stem-loop tag sequences, we demonstrate that even mixtures of multiple adjacent or overlapping alleles in the same locus of EGFR/KRAS/BRAF mutations can be specifically amplified in the same reaction and distinguished by amplicon size differentiation in a single lane of an agarose gel. Moreover, using primer sets specific for methylated and converted DNA, and those specific for unmethylated and converted DNA, we demonstrate that EGFR/KRAS/BRAF mutations and their associated promoter methylation status can be amplified from bisulfite-treated genomic DNA of colon cancer cell lines in a single reaction. In conclusion, the Applied Biosystems Platinum® Multiplex PCR Master Mix and the stem-loop primer system are a “best in class” solution to simultaneous amplification of various types of genetic and epigenetic tumor markers of multiple targets for high throughput genotyping and aberrant methylation profiling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3070. doi:10.1158/1538-7445.AM2011-3070