bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1094: Transcriptional activation by pRB/p107 and coordination with SWI/SNF
Authors: Flowers Stephen , Beck George R. , Moran Elizabeth
Published: February 2011

The retinoblastoma susceptibility gene, Rb1, is closely linked with osteosarcoma as well as retinoblastoma. The basis of such ties between individual susceptibility genes and tumors of specific tissue origin is a central question in cancer biology. Functional analysis of pRB and the closely related family members, p107 and p130, has tended to focus on repression of proliferation, but genetic evidence from our lab and others indicates an active requirement for pRB in osteoblast differentiation that might relate more directly with osteosarcoma susceptibility. Our previous studies linked pRB with activation of the early osteoblast marker, alkaline phosphatase (the product of the Akp2 gene). Here we have investigated the functional relationships between pRB and the SWI/SNF complex in Akp2 activation. Chromatin immunoprecipitation (ChIP) assays reveal the Akp2 promoter is targeted directly by all three pRB family members. Only p130 occupies the promoter prior to induction, while pRB and p107 target Akp2 directly during activation, and concurrent with association of the global co-activator p300. Akp2 is also a direct target of SWI/SNF chromatin remodeling complexes. These ATPase-powered complexes help orchestrate the programmatic shifts in gene expression necessary for progression from a self-renewing precursor state to a state of terminal differentiation. In osteoblasts, SWI/SNF complexes that repress osteogenic gene expression in order to maintain the precursor state are characterized by inclusion of the BRM ATPase. The alternative ATPase, BRG1, characterizes complexes that activate osteogenic genes during terminal differentiation. Thus, the onset of osteoblast differentiation is marked by a switch from BRM to BRG1 complexes on the Akp2 promoter. Our analysis shows that the BRM to BRG1 switch is accomplished in a precise two-step mechanism, with dissociation of BRM-containing SWI/SNF dependent on p300, and association of BRG1-containing SWI/SNF dependent on pRB/p107. Binding of RNA polymerase-II is dependent in turn on BRG1. Thus, in contrast to their widely studied role in cell cycle repression, pRB/p107 play a directly activating role in osteogenic gene expression, specifically in recruitment of the activating SWI/SNF complex.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1094.
Abstract 1098: Cys141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines
Authors: Yusuf Mohammad A. , Srivenugopal Kalkunte S.
Published: February 2011

Purpose: We previously described that human p53 is functionally inactivated by S-glutathionylation at Cys 141 during oxidative and genotoxic stresses (Biochemistry 46, 7765-7780, 2007). To study the dynamics of this posttranslational modification in human cancers and normal tissues, polyclonal antibodies specifically recognizing the thiolated p53 were generated and characterized.

Experimental Design: An 18 residue p53 peptide harboring a mixed disulfide with GSH at Cys141 was synthesized, and antibodies were raised in 5 rabbits. After initial assessment of the sera for the presence of specific (p53-glut) antibodies, it was affinity purified. The antibodies were tested in ELISA, immunoblotting and immunohistochemistry applications using human cancer cell lines and tumor tissue microarrays.

Results: The p53-glut antibodies recognized the antigenic peptide but not the unmodified counterpart, with high sensitivity in ELISA. In immunoblot analyses, the purified antibodies did not react with unmodified p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or small molecule ethanediol-modified rp53 only under non-reducing conditions. Western blotting and immunocytochemistry in HCT116 cells showed low basal levels of thiolated p53 in untreated cells which increased markedly after acute oxidative stress induced by H2O2, diamide and DNA damage by cisplatin or doxorubicin. The glut-p53 decreased sharply after passage of cells to oxidant-free media. The mutant p53 present in HT29 cells was also recognized. In human prostatatic tissues, the apparently normal prostate adjacent to the tumor and most prostate cancers showed abundant presence of glut-p53 in luminal epithelium, a site well known to generate reactive oxygen species, hydrogen peroxide, and lack antioxidant enzymes such as GST-pi. The melanoma and colon cancer were also positive while gastric cancers were negative. Further, the glut-p53 was degraded faster than unmodified p53 in the presence of rabbit reticulocyte lysates.

Conclusions: Our findings provide first evidence for the presence of glut-p53 in human cells and cancer specimens. Glut-p53 found in prostate tissues may reflect the chronic oxidative stress and an alternative route to functional inactivation of p53. This is a first description of thiolation-specific antibodies for any protein. The glut-p53 antibodies should provide a valuable tool to study p53 inactivation in sustained redox imbalance states found in cancer and degenerative diseases, as well as the therapy-induced negative regulation of p53.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1098.
Abstract 1105: Dysregulated ΔNp63α displaces p73 from the promoter of the tumor suppressor gene maspin in keratinocytes
Authors: King Kathryn E. , Muraleedharan Deepti , Ponnamperuma Roshini M. , Gerdes Michael , Weinberg Wendy C.
Published: February 2011

The p53 homologue p63 is critical for normal epidermal development. While overexpression of p63, in particular ΔNp63α, has been reported in many squamous cell cancers including those of the skin, the role of p63 in cancer pathogenesis remains unclear. We have previously shown that elevated levels of ΔNp63α aberrantly maintain keratinocyte proliferation under differentiating conditions. We performed microarray analyses of primary murine epidermal keratinocytes overexpressing adenoviral (Ad)-driven ΔNp63α vs. β-gal to identify target genes impacted by dysregulated ΔNp63α. Our studies revealed a coordinated downregulation of RNA transcripts for multiple protease inhibitors, including two members of the serine protease inhibitor (serpin) family, maspin (serpinB5) and plasminogen activator inhibitor-2, PAI-2 (serpinB2), in addition to the metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-3 (TIMP-3). Downregulation of all three protease inhibitors was confirmed by RT-PCR and examined further by western analysis. TIMP-3 protein was detected in keratinocyte conditioned medium but not in whole cell lysates, supporting a primarily extracellular function for TIMP-3. PAI-2 and maspin were detected in both whole cell lysates and conditioned medium, consistent with both intracellular and extracellular roles for these proteins. Secreted levels of TIMP-3 and PAI-2 declined in Ad-ΔNp63α-cultures compared to Ad-β-gal-cultures, whereas maspin levels remained stable. Intracellular expression of both PAI-2 and maspin was negatively regulated by ΔNp63α. To investigate the mechanism by which ΔNp63α down-regulates maspin, we performed electrophoretic mobility shift assays (EMSAs) using a known p53 consensus DNA binding sequence in the maspin promoter. An endogenous complex was seen by EMSA in normal keratinocytes that was disrupted in the presence of increasing levels of ΔNp63α. Supershift analysis revealed that this endogenous complex was interrupted by the addition of a p73 antibody, but no supershift or block was seen with antibodies to p63 or p53. EMSA analysis of nuclear extracts prepared from p73(-/-) keratinocytes confirmed that p73 is required for this complex formation. The current data suggest additional roles for ΔNp63α in cancer pathogenesis by disrupting p73 transcriptional activity to down-modulate protease inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1105.
Abstract 1108: The Drosophila Caliban suppresses tumor formation through both DNA damage repair and apoptosis
Authors: Bi Xiaolin , Wang Zhe , Mortin Mark A. , Wang Yajie , Stultz Brian , Hursh Deborah A.
Published: February 2011

The tumor suppressor gene sdccag1 is down-regulated in human colon cancer cell lines, its Drosophila homolog caliban was screened using a functional HDA domain of homeobox gene prospero as bait. In our previous work, we had suggested Caliban is a bipartite nuclear export mediator, working through the Exportin pathway, and itself can shuttle between cytoplasm and nucleus. To study its tumor suppression functions, we generated knock-out Drosophila Melanogaster of caliban by gene targeting strategy. The caliban knock-out flies are viable and fertile, similar as tumor suppressor gene Dmp53 knock-out flies. While caliban null flies have retarded growth by three days, this delayed growth was rescued by Dmp53 mutation. The caliban flies are sensitive to irradiation, and its DNA damage sensitivity depends on p53. High dosage treatment with irradiation causes tumor formation in caliban null flies, while atm and caliban double mutant flies have autonomous tumor formation, which is similar as atm p53 double mutant. The tumor suppression function of caliban was also studied in an in vivo mouse model where lung tumor cells A549 were implanted subcutaneously into athymic nude mice, we observed the greatly reduced tumor size in caliban expressing A549 cells. These preliminary results suggest caliban may compensate with p53 to suppress the tumor predisposition through both DNA damage repair and apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1108.
Abstract 1111: Enhancer of Zeste homologue 2 (EZH2) regulates BRCA1 transiently nuclear export during the cell cycle, and induces tetraploidy of breast cells through AKT1
Authors: Gonzalez Maria E. , Li Xin , Toy Kathy , Prie Mattew Du , Ventura Alejandra , Kruger Heather , Kleer Celina G.
Published: February 2011

EZH2 is overexpressed in breast carcinomas with high propensity to metastasize. EZH2 is an oncogene essential to promote proliferation and capable to trigger neoplastic conversion in the breast. We have reported that EZH2 promotes proliferation of breast cancer through downregulation of BRCA1 protein, but the mechanism is unknown. BRCA1 shuttles between the cytoplasm and nucleus to maintain genomic stability. AKT1 up regulation disrupts BRCA1 function by altering its nuclear localization and inducing genomic instability. We hypothesize that EZH2 regulates nuclear-cytoplasmic shuttling of BRCA1 in breast cells through AKT1.

Methods: We developed two cell culture models: an inducible EZH2 up regulation system in MCF10A benign mammary cells and a lentivirus-mediated shRNA interference targeting EZH2 model in CAL51 breast cancer cells (tetraploid, hormone receptor negative, and with intact p53 and BRCA1 genes). Our recently developed EZH2 transgenic mouse model was analyzed to determine the in vivo relevance of our in vitro data obtained by Western blots, immunofluorescence, proliferation, and FACS.

Results: EZH2 shRNA knock-down in CAL51 cells increased BRCA1 nuclear localization in early S phase, decreased cell growth by delaying cell cycle progression, and tetraploidy. EZH2 overexpression in MCF10A cells decreased BRCA1 nuclear localization, increased proliferation and caused mitotic spindle defects and tetraploidy. EZH2 overexpression increased total and phoshporylated AKT1 at serine 473. Specific pharmacologic inhibition of AKT reverted the effect of EZH2 on BRCA1 nuclear localization, proliferation, mitotic defects and tetraploidy. We also found that EZH2 directly interacts with AKT1 protein. Mammary glands of EZH2 transgenic mice show up-regulation of AKT1 and a decreased in nuclear BRCA1 protein compared to wild type mice.

Conclusion: EZH2 regulates the nuclear-cytoplasmic shuttling of BRCA1 protein and influences BRCA1 functions on proliferation, mitotic spindle control, and maintenance of genomic stability in benign breast cells and in breast cancer cells. The regulation of BRCA1 localization and function by EZH2 requires AKT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1111.
Abstract 1117: Characterization of BAT1 (UAP56) interaction with BARD1
Authors: Carvalho Renato S. , Nepomuceno Thales C. , Sweet Michael , Monteiro Alvaro N. , Carvalho Marcelo A.
Published: February 2011

BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid screen as a binding partner of BRCA1. The functional heterodimer BRCA1-BARD1 is required for several of the cellular and tumour-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair (DDR) pathway, transcription, cell cycle control, and ubiquitination. BARD1 also contains two tandem BRCT repeats at its C-terminus, a region shown to bind serine-phosphorylated peptides in vitro. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a yeast two hybrid (Y2H) screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for residues 308-418 of BAT1 (UAP56, C-terminal region [OMIM 142560]). UAP56 is a member of DEAD box family of ATP-dependent RNA helicases. Several studies indicate that UAP56 protein is not only essential in pre-mRNA splicing but is also important in mRNA nuclear export and cytoplasmic mRNA localization. We confirmed BAT1-BARD1 interaction by conducting co-IP analysis of ectopic expressed BARD1 and BAT1 in HEK293T cells. We are using a Y2H system approach to determine the minimal region of interaction between BARD1 and BAT1; in parallel, we are also checking for the presence of a multicomponent complex, investigating the presence of BRCA1. In order to assess the biological significance of the interaction between BARD1 and BAT1 we will determine whether this interaction is important for the activation of, or recovery from, the G2/M cell cycle checkpoint after IR and also investigate its putative involvement in p53-dependent apoptosis pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1117.
Abstract 112: Concurrent analysis between copy number variation and gene expression of non-smoking lung cancer females in Taiwan
Authors: Chang Jung-Chih , Lu Tzu-Pin , Chen Pei-Chun , Lee Jang-Ming , Hsu Chung-Ping , Tsai Mong-Hsun , Chuang Eric Y. , Lai Liang-Chuan , Hsiao Chuhsing K.
Published: February 2011

Although smoking has been proved to be an important factor for lung cancer, the etiology and molecular mechanisms of non-smoking female lung cancer remain unclear. We collected paired normal and tumor lung tissues from 44 female non-smoking lung cancer patients. Copy number variation and Gene expression profiles were analyzed by Affymetrix U133plus 2.0 expression arrays and Affymetrix SNP 6.0 single nucleotide polymorphism (SNP) arrays. The data from two platforms were integrated for concurrent analyses in order to elucidate the putative molecular mechanisms. We first identified the probes located at the same region of gene expression and copy number variation (CNV). Next, we calculated the Pearson correlation coefficient to assess the association between log intensity values of mRNA expression and the corresponding averaged intensity values of SNP probes in both tumor and normal tissues. Genes were then selected based on the following two criteria: (1) the difference in expression-SNP correlations between normal and tumor tissues > 0.7, and (2) the absolute expression-SNP correlation of either normal or tumor >0.3. The selected genes were further analyzed by Ingenuity Pathway Analysis (IPA) to explore the related pathways and networks. Results of IPA showed that mitochondrial dysfunction, IGF-1 signaling, and cell cycle: G1/S checkpoint regulation pathways were significantly dysregulated in tumor tissues. The associations between lung cancer and genomic alternations in these enriched pathways have been reported previously, such as overexpression of IGF1R in IGF-1 signaling pathway might resist EGFR inhibitors used in lung cancer treatment. In summary, with the integrated data of copy number variation and expression, the concurrent analyses help to dissect lung carcinogenesis of non-smoking lung cancer females in Taiwan.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 112.
Abstract 1120: Low prevalence of TP53 mutations in pediatric rhabdomyosarcoma
Authors: Ognjanovic Simona , Martel-Planche Ghyslaine , Olivier Magali , Hainaut Pierre
Published: February 2011

The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. Mutations in TP53 in rhabdomyosarcoma (RMS) have been associated with aggressive tumors and poor prognosis, however, reported prevalence of mutations in these tumors varied with smaller series reporting larger prevalence. Apart from gene mutations, p53 function can also be attenuated by overexpression of MDM2. Most recent report suggested extremely low prevalence of TP53 mutations and MDM2 amplifications (Taylor et al., 2000). We had 40 embryonal and 40 alveolar RMS tumor samples available for TP53 mutations/MDM2 amplification analyses, the largest series analyzed thus far.

Half of the available samples were histopathology slides and the other half were frozen tumor tissue. The average age of slides was 13 years, therefore only 24 out of 40 slide DNA samples were of sufficient quality/quantity to complete direct sequencing of TP53 exons 4-9, limiting our results to 64 samples. MDM2 amplification was analyzed by differential PCR using dopamine gene as control. TP53 sequencing detected only one single mutation, p. D259Y, and six polymorphisms (Table 1).Table 1.Mutations and polymorphisms in TP53 gene in 64 pediatric embryonal and alveolar RMS. DiagnosisAgeGenderRacePrimary siteStageGene Variation*Protein ChangeMutationsERMS1MWhiteRight thighN/Ag.13412G>Tp. D259YPolymorphismsERMS0MBlackRight ankleN/Ag.14077T>CN/A ERMS4FBlackBladder2g.12708A>Gp. R213R ARMS4MAsianButtockN/Ag.12708A>Gp. R213R ARMS6MWhiteForearmN/Ag.12708A>Gp. R213R ERMS6FWhiteUterusN/Ag.12708A>Gp. R213R ARMS9FWhiteParotid mass1g.11370C>Tp. P47S* nucleotide number based on GenBank sequence NC_000017.9

No distinct pattern between TP53 mutation/polymorphisms was associated with any RMS subtype. Likewise, no MDM2 amplifications were observed.

In conclusion, we confirm the extremely low prevalence of TP53 mutations/MDM2 amplifications in pediatric RMS (1.25% and 0% respectively). The possible inactivation of p53 function by other mechanisms thus remains to be elucidated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1120.
Abstract 1122: UVB-induced PTEN down-regulation and its association with skin carcinogenesis
Authors: Ming Mei , Han Weinong , Shea Christopher R. , Soltani Keyoumars , Maddox Jessica , Freeman Diane M. , He Yu-Ying
Published: February 2011

Non-melanoma skin cancer including squamous cell carcinoma (SCC) is the most common cancer in the United States and ultraviolet (UV) radiation in sunlight is a major environmental trigger for skin carcinogenesis. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a proven critical tumor suppressor. We found that UVB down-regulates PTEN in primary human keratinocytes, human HaCaT keratinocytes and mouse skin, and the ERK/AKT/PTEN axis may form a positive feedback loop following UVB irradiation. We also examined PTEN levels in different types of skin cancer that are associated with chronic sun exposure, including actinic keratoses (AK, premalignant lesion), keratocanthoma (KA), squamous cell carcinoma (SCC) in situ (SCC is), SCC invasive (SCC iv). As compared with normal skin, PTEN expression is significantly down-regulated in human skin cancer with these different diagnoses. In addition, PTEN down-regulation is significantly associated with solar UV damage and age of patients. Our findings indicate that UVB radiation suppresses PTEN expression in human skin cancer and this down-regulation may play critical roles in the development and progression of human skin cancer.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1122.
Abstract 1123: PTEN loss increases efficiency of breast tumor metastasis
Authors: Vitolo Michele I. , Thompson Keyata , Whipple-Bettes Rebecca A. , Matrone Michael A. , Yoon Jennifer R. , Balzer Eric M. , Cho Edward H. , Martin Stuart S.
Published: February 2011

Breast cancer is the most frequent malignancy in women, and although many breast cancers are curable via surgery, approximately one quarter maintain a latent and insidious characteristic of slow growth with early metastasis. The loss of the tumor suppressor PTEN has been associated with breast cancer stage, lymph node status, and disease-related death, and the high rate of loss in primary tumors suggests a potential role in initiation and/or progression of the disease. However, specific cellular alterations in human breast epithelium controlled by PTEN inactivation, which lead to an increased metastatic phenotype, remain poorly defined. We have determined that heterozygous and homozygous loss of PTEN in non-tumorigenic mammary epithelial cells (MECs) was insufficient to promote anchorage-independent growth. However, MECs with PTEN loss maintained elevated activation of the PI3K/Akt and MAPK pathways and apoptotic resistance to cell rounding and matrix detatchment. We have also recently determined that PTEN expression loss leads to the production of long, dynamic, tubulin-based membrane protrusions upon detachment, which are increased in frequency, number and length per cell as compared to their isogenic, PTEN-expressing parental counterparts. These novel structures, termed microtentacles (McTNs), are structurally distinct from classical actin-based extensions of adherent cells, persist for days in breast tumor lines that are resistant to anoikis, and aid in the reattachment to matrix or cell monolayers. Therefore, the combination of apoptotic resistance and enhanced McTN formation due to PTEN loss may have important consequences for facilitating tumor cell extravasation and efficient adherence in metastatic sites.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1123.
Abstract 1124: Characterization of a PTEN C2 loop mutation in a human breast cancer cell line
Authors: Chan Andrew M. , Singh Gobind , Odriozola Leticia , Kennedy Colin
Published: February 2011

PTEN is a major tumor suppressor gene inactivated in over 30% of human cancer. It encodes a lipid phosphatase with specificity towards phosphatidylinositol (3,4,5) triphosphate (PIP3). PTEN phosphatase activity is crucial to its ability to control cell size, proliferation, motility and survival. The role of PTEN in human breast cancer is well-established. Although the frequency of PTEN mutation in human breast cancer is low (5-10%), a significant fraction of advanced breast cancer has reduced PTEN expression. Furthermore, germ line mutation in PTEN gene constitutes the genetic basis of Cowden's Disease (CD) Syndrome. CD is a harmatoma syndrome with patients displaying aberrant overgrowth of multiple tissue types. Individuals are at a greater risk of developing malignancy of the breast, colon, skin and thyroid.

Our laboratory has identified a unique mutation in the C2 loop of PTEN from a human metastatic breast carcinoma cell line, MDA-MB-453. A G to A change substitutes the glutamate (GAG) at codon 307 (E307) to a lysine (AAA). The PTEN C2 loop (aa282-312) is a region on the cytosolic side of the C2 membrane-binding domain. Mono-ubiquitination at K289 within the C2 loop has been shown to promote nuclear translocation. More importantly, unlike the wild-type protein, the E307K mutant is almost undetectable in the nuclear compartment of MDA-MB-453. Biochemically, PTEN E307K mutant displayed similar phosphatase activity towards soluble lipid substrates. More importantly, PTEN E307K displays a striking ability to form an affinity complex with the p53 tumor suppressor. Consistent with this finding, the expression of E307K exerts chemoprotective effect against cisplatinum induced cell death.

Based on this preliminary data, we hypothesize that the negatively-charged glutamate residues of PTEN C2 loop play regulatory roles in nuclear translocation and p53-binding. Mutating those residues to lysine creates cryptic ubiquitination sites that subvert the natural ubiquitination at K289, and thus impedes nuclear translocation.

Funding: DOD - DAMD17-03-1-0682 (AMC); NCI - RO1CA095063 (AMC), T32CA078207 (GS); Midwest Athletes against Childhood Cancer (AMC); Advancing Healthy Wisconsin Funds (AMC)

Acknowledgement: Dr. Xuejun Jiang (Memorial Sloan Kettering Cancer Center, NY) and Dr. Zhen-Qiang Pan (Mount Sinai School of Medicine, NY) for providing the NEDD4-1 and ubiquitination reagents

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1124.
Abstract 1127: Dysregulation of tumor suppress gene LKB1 in pancreatic cancer
Authors: Wei Chongjuan , Zhang Nianxiang , Liu Qian , Amos Christopher , Frazier Marsha
Published: February 2011

Pancreatic cancer is the fourth leading causes of cancer-related death in USA and the most common seen neoplsams in Peutz-Jeghers syndrome (PJS). PJS is an autosomal dominant disorder characterized by gastrointestinal hamartoma and predisposition to cancer. Patients with PJS have a 100-fold higher risk for development of pancreatic cancer than the general population. Tumor suppressor LKB1 is the causative gene for PJS. LKB1 mutations have also been observed in a significant proportion of sporadic pancreatic cancer, suggesting a role for LKB1 in pancreatic cancer although the underlying mechanism has not been elucidated yet. To evaluate the role of LKB1 in pancreatic tumorigenesis, we extracted RNA from our unique collection of fifty-three tumor/normal pairs of snap frozen pancreatic cancer and adjacent normal tissue from the same patient. RT-PCR results showed that LKB1 is downregulated in ∼74% of the cancer tissue compared to adjacent normal pair, and in ∼40% of pairs the downregaulation is greater than 2 folds. To confirm our result, we analyzed the publically available gene expression microarray data on 36 pancreatic cancer samples and 12 normal controls. We observed downregulation of LKB1 in these data and the difference in LKB1 expression levels in pancreatic cancer vs normal was statistically significant (p=0.0003). LKB1 has been reported to negatively regulate mTOR signaling. Therefore, we further analyzed whether there was correlation between the expression of LKB1 and key protein in mTOR signaling pathway. Our results showed that the expression of LKB1 is inversely correlated with that of EIF4A and EIF4E, which are translation initiation factors. These findings suggest that dysregulation of LKB1 and its downstream signaling pathway may play an important role in pancreatic tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1127.
Abstract 1136: Detection and characterization of circulating tumor cells in localized and metastatic prostate cancer patients using CTC-Chip microfluidic technology
Authors: Nagrath Sunitha , Stott Shannon L. , Lee Richard J. , Yu Min , Ulkus Lindsey L. , Iafrate John A. , Smith Mathew R. , Tompkins Ronald G. , Sequist Lecia V. , Haber Daniel A. , Maheswaran Shyamala , Toner Mehmet
Published: February 2011

Despite recent molecular advances, the treatment of localized and metastatic prostate cancer has changed little in recent decades and it remains a major cause of cancer deaths in men. Localized prostate cancer may be either indolent or invasive, without reliable distinguishing markers, and the absence of tumor tissue from patients with metastatic bone disease has hampered understanding of acquired resistance to primary hormonal therapy. Given the predominant hematogenous spread of prostate cancer, circulating tumor cells (CTCs) may provide an indicator of vascular invasiveness, as well as a source of tumor specimens for molecular analysis, but available technologies have been limited by relatively low sensitivity. Huge strides in engineering at smaller scales have lead to the tremendous progress in microfabricated devices to facilitate both applied and basic research. Applying a multi-disciplinary approach, we have developed a microfluidic technology “CTC-Chip” for diagnosis and monitoring of non-hematological cancers. Using the CTC-Chip, we were able to isolate CTCs in patients with both localized and metastatic prostate cancer. In patients with advanced cancer, CTCs were present at a frequency of 34 CTCs/mL (range 14-653), with a profound decline in numbers during response to hormone withdrawal therapy. Molecular markers, including the characteristic TMPRSS2-ERG translocation, are readily identified in CTCs. In patients with early stage, resectable prostate cancer, we have monitored CTC load in circulation following surgical resection of the primary tumor. CTCs were examined the day following surgical resection, at postoperative day 9, and at greater than 3 months, coincident with scheduled postoperative clinical visits. Longitudinal monitoring of CTCs revealed all CTC-positive cases showed disappearance of CTCs following prostate resection, with some having an immediate decline and others having a delayed (>24 hours) response. No evident differences in tumor pathology or operative procedure distinguished patients with a rapid versus delayed postoperative CTC decline. Dual staining of captured CTCs for PSA and Ki67 indicated a broad range in proliferative index of CTCs among patients with different clinical courses. Thus, the quantitative isolation and analysis of CTCs in both localized and metastatic prostate cancer provides novel insights into early hematogenous dissemination and enables molecular analyses of treatment-responsive and -resistant disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1136.
Abstract 1139: Complete characterization of prostate cancer genomes by massively parallel sequencing
Authors: Berger Michael F. , Lawrence Michael S. , Cibulskis Kristian , Pflueger Dorothee , Demichelis Francesca , Sougnez Carrie , Onofrio Robert C. , Ambrogio Lauren , Fennell Timothy , Parkin Melissa , Carter Scott L. , Saksena Gordon , Sivachenko Andrey , Voet Douglas , Wilkinson Jane , Fisher Sheila , Winckler Wendy , Ardlie Kristin , Chant John , Baldwin Jennifer , Gerstein Mark , Golub Todd R. , Meyerson Matthew , Tewari Ashutosh K. , Gabriel Stacey B. , Lander Eric S. , Getz Gad , Rubin Mark A. , Garraway Levi A.
Published: February 2011

Prostate cancer is the most common type of cancer diagnosed among men in the United States, accounting for 200,000 new cases and 27,000 deaths per year. Prior genetic studies have shown that chromosomal rearrangements comprise a major mechanism of oncogene activation in prostate cancer. For example, androgen-regulated gene fusions involving ETS family transcription factors are present in the majority of prostate cancers, yet the full repertoire of genomic alterations driving prostate carcinogenesis and progression remains unknown. Toward this end, recent technological advances have made it possible to characterize the full complement of somatic mutations in a single tumor through whole genome sequencing.

We are using massively parallel sequencing technology to characterize the complete genomes of several primary prostate adenocarcinomas at >30x coverage. All samples are high-grade primary tumors (Gleason grade 7 to 9) and include cases with and without known ETS family translocations. For each tumor, we are also obtaining >30x sequence coverage of matched normal DNA from blood of these same patients in order to determine the somatic component of the overall variation we observe. Our results indicate that translocations and other chromosomal rearrangements occur frequently in prostate cancer, at a rate of >100 per genome. Further, we have discovered many nonsynonymous sequence mutations (point mutations and indels) in each tumor, some of which may represent novel candidate drivers of tumor progression. The overall rate of somatic point mutations is approximately 1 per Megabase. Integrated analysis of all genomes reveals both recurrent and private alterations. Together, these results illuminate potential avenues for target discovery and demonstrate the unparalleled value in performing complete genome sequencing in this malignancy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1139.
Abstract 1142: Development and validation of a multiplexed PCR-coupled liquid bead array for gene expression in circulating tumor cells
Authors: Markou Athina N. , Strati Areti D. , Malamos Nikolaos , Georgoulias Vasileios , Lianidou Evi S.
Published: February 2011

INTRODUCTION: The aim of our study was to develop and validate a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in Circulating Tumor Cells (CTCs), and application of this assay in peripheral blood samples of breast cancer patients.

PATIENTS AND METHODS: We designed a novel multiplexed PCR-coupled liquid bead array that consist of the following steps: a) in silico designed gene-specific primers and capture probes for CK-19, HER-2, Mammaglobin (hMAM), MAGE-A3, TWIST-1 and PBGD (as a control gene), b) RNA isolation from immunomagnetically enriched CTCs, c) multiplex PCR, d) sequence hybridization array, e) detection in the Luminex platform. We performed extensive optimization experiments using the SKBR3 and MDA-MB-231 cell lines as positive controls, to maximize analytical sensitivity and specificity and we further validated the performance of this array in respect to cross reactivity and intra and inter-precision. Finally, we applied the developed methodology in peripheral blood samples of 37 patients with operable breast cancer, 25 patients with verified metastasis and 17 healthy individuals.

RESULTS: The analytical performance of the developed array was evaluated in tumor cell lines in respect to analytical sensitivity and specificity. Cross reaction studies were performed for each gene target in the presence of all other targets. The assay is highly specific for each gene in complex multiplexed formats, since the discriminatory power of fluorescent bead sets is unique to specifically detect by sequence hybridization the presence of individual gene specific PCR products. The developed liquid bead-based array is highly sensitive, since it can detect the expression of each individual gene at one SKBR3 cell level. The validation of the array included within day and between-days precision studies. None of the genes tested was detected in the CTC fraction of healthy donors while in patients with verified metastasis, CK-19 was detected in 45%, HER-2 in 20%, MAGE-A3 in 30%, hMAM in 15% and TWIST-1 in 20%. In operable breast cancer patients, CK-19 was detected in 13.5%, HER-2 in 5%, TWIST-1 in 16.2%, MAGE-A3 in 37.8% while hMAM was not detected in any sample.

CONCLUSIONS: Our results show that individual gene expression can be readily measured in CTCs using a bead-based platform. This may form an efficient basis for a multiplex approach to measure multiple genes (up to 100) in the same sample, thus saving sample volume and reducing the total cost and time of analysis. This is the first time that the Luminex technology is used for gene expression studies in CTCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1142.
Abstract 1147: High-throughput nanofluidic PCR-based preparation of 454 sequencing libraries for identifying mutations in EGFR and MET
Authors: Wang Jun , Anderson Megan J. , Kaper Fiona , Stewart Claudia , Soppet Daniel , Bere Bill , Hartley Todd , Chen Peilin , Lin Min , Pieprzyk Martin , Jones Robert C. , May Andrew P.
Published: February 2011

The epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) play important roles in cancer development and are promising targets for anticancer therapies. Mutations in the EGFR and MET genes are associated with multiple types of cancer and the ability to detect variants in these genes is crucial for a better understanding of disease progression and response to targeted therapeutics. Next-generation sequencers (e.g. 454, Illumina) hold great potential for detecting disease-specific alterations within patient populations for a given cancer type. However, current approaches for targeted resequencing of specific genetic regions are not well suited to studying the hundreds or thousands of patient samples required to assign significance to mutations.

We have developed a novel nanofluidic platform, the Access Array™ system, which enables the robust, simultaneous amplification of 48 PCR products (amplicon) from 48 samples in parallel using only 50ng input DNA per sample. In addition, we have developed a primer design strategy that incorporates sample-specific barcodes and 454 sequencing adaptors into each amplicon, removing the need for library preparation before sequencing. We will present sequencing data collected from amplicon libraries prepared using the Access Array system to amplify 48 exon regions of the EGFR and MET genes from the NCI-60 cell lines. Sequencing data from these libraries demonstrates excellent uniformity in representation between amplicons and across samples. We will present data mapping mutations within these genes across the NCI-60, and discuss the use of Access Array enrichment panels in monitoring mutations across large patient populations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1147.
Abstract 1154: Direct detection of DNA methylation and mutagenic damage through single-molecule, real-time (SMRTTM) DNA sequencing
Authors: Flusberg Benjamin , Webster Dale , Travers Kevin , Olivares Eric , Korlach Jonas , Turner Stephen
Published: February 2011

Changes in genomic methylation patterns are often associated with cancer processes. The development of new, high-throughput techniques for mapping aberrant methylation patterns, along with various forms of DNA damage, will be important for advancing cancer research. Single-molecule real-time (SMRTTM) sequencing yields a rich set of information about nucleic acid structure that allows direct detection of various forms of modified nucleotides, including 5-methylcytosine, 5-hydroxymethylcytosine, N6-methyladenosine, and 8-oxoguanosine. We have studied DNA templates with a range of CpG methylation patterns, and we describe how this approach, in combination with circular consensus sequencing of individual DNA molecules, can be used for base-pair resolution identification of epigenetic modifications. Unlike current bisulfite-sequencing techniques, which are limited by short read lengths and by the reduction in genomic complexity, our method will enable mapping of methylation patterns within even highly repetitive genomic regions. Overall, these results highlight the potential applications of the SMRTTM technology to high-throughput sequencing of methylation and mutagenic DNA lesions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1154.
Abstract 1162: RNA expression analysis of rare tumor cells by laser-mediated lysis
Authors: Kamme Fredrik , Bright Gary
Published: February 2011

Molecular analysis of live rare cells, such as circulating tumor cells (CTCs) is challenging. Current strategies include antibody-mediated capture and size selection and are limited in the purity and recovery of the cells of interest. We have developed a novel approach for rare cell analysis based on the specific lysis of targeted cells in a background of a vast majority of other cell using a precisely targeted laser pulse. Instantly lysed cells release their contents into the medium, including mRNA, providing a method to analyze mRNA content in selected cells. Using a high throughput plate cytometer equipped with lasers for in situ cell manipulation (LEAP Cell Processing and Purification Workstation), we have analyzed RNA from individual tumor cells in a model of CTCs consisting of carcinoma cells spiked into human PBMCs. A 5-gene panel was measured by RT-PCR from single tumor cells and was detected in 94% of the cases with 0% false positives. RNA analysis of single tumor cells via laser-mediated lysis represents a new principle for the analysis of rare cells, as the specificity of analysis is dependent on imaging and laser targeting only and free of any limitations imposed by an antibody- or size-dependent purification strategy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1162.
Abstract 1167: The GNAS1 T393C polymorphism is a predictor of clinical outcome in esophageal cancer
Authors: Effenberger Katharina E. , Musici Safije , Yekebas Emre F. , Kutup Asad , Kamradt Till , Mina Sormeh , Kalinin Viacheslav , Brandt Burkhard , Pantel Klaus , Izbicki Jakob R. , Vashist Yogesh K.
Published: February 2011

Background: There is a paucity of prognostic markers in esophageal cancer (EC). Genetic variations determined in the germline DNA of cancer patients may serve as important prognostic factors of clinical outcome since genomic DNA remains unbiased by instability occurring in tumor DNA and is easily accessible in the peripheral blood. G-protein α subunit (Gαs) polymorphism has been identified as a prognostic marker of tumor recurrence and survival in various tumor entities. We genotyped the single nucleotide T393C polymorphism in the GNAS1 gene encoding Gαs, and focused on three different genotypes, TT, TC, and CC. The aim of this study was to evaluate the potential prognostic value of the GNAS1 polymorphism in EC.

Methods: Genomic DNA was extracted from peripheral blood leucocytes of 190 patients who underwent complete resection for esophageal cancer and did not receive any neoadjuvant or adjuvant therapy. The GNAS1 genotypes were correlated with clinicopathological parameters, tumor cell dissemination in bone marrow and clinical outcome.

Results: The TT and TC genotypes correlated with advanced disease, predominantly characterized by the presence of lymph node metastases (p<0.001) and tumor cell dissemination (p=0.01). In line with this, significantly more TT and TC genotype patients displayed disseminated tumor cells in bone marrow compared to the CC genotype group (p=0.01). Accordingly, the disease recurrence rate was higher in TT and TC than in CC patients. Kaplan-Meier estimates for disease-free (DFS) and overall survival (OS) displayed better outcome in CC compared to TT and TC genotypes. Furthermore, in multivariate Cox proportional hazard analysis, GNAS1 polymorphism independently predicted disease progression with the CC group presenting the best outcome (OS: p=0.001, DFS p=0.002).

Conclusion: The GNAS1 polymorphism was identified as a strong independent prognostic factor for DFS and OS in EC patients supporting a functional role of Gαs in this tumor entity. Preoperative determination of the GNAS1 polymorphism will allow allocation of patients into different risk profiles as well as stratification into individual therapy regimen.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1167.