bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4823: Single cell analysis of head and neck squamous cell carcinoma cells isolated from primary tumors and matched metastases
Authors: Sproll Karl C. , Behrens Bianca , Hollinger Andrea , Baldus Stephan E. , Kübler Norbert R. , Knoefel Wolfram T. , Brakenhoff Ruud H. , Stoecklein Nikolas H.
Published: June 2012

Background: Genetic heterogeneity is a hallmark of cancer and significant evolution occurs locally and at metastatic sites with disease progression. This heterogeneity might contribute significantly to therapy resistance. Since systematic in depth analyses are lacking in head and neck squamous cell carcinomas (HNSCC), we established and tested a protocol that enables comprehensive single cell analyses of cancer cells from primary tumors and metastasis.

Material and Methods: From matched primary tumors and lymph node metastases of two HNSCC patients, single cell suspensions were generated and sedimented on positively charged glass slides at low density. Epithelial tumor cells were identified with double-immunofluorescence staining for CD44v6 and CK5/14. Twelve to 14 double-positive cells were isolated via micromanipulation from each tumor and an adapter-linker PCR was used for whole genome amplification of single cells. Sequencing of TP53 was performed on genomic DNA from primary tumors as well on single cell amplification products. An allele-drop-out experiment was performed to control for artificial allelic loss. Fluorescence-in-situ-hybridization (FISH) was used to determine the copy number of chromosome 17 and TP53-gene loci, respectively.

Results: Two different heterozygous TP53 mutations were detected in both cases. On the single cell level, we observed an unexpected variation of wild-types, heterozygous mutations and homozygous mutations on the investigated mutation sites. This mutational distribution was significantly different from the allele-drop-out experiment (drop-out rate: 8%). In one we found an expansion of mutated clones in metastasis, and in the other patient a transmission of the genetic changes between the primary tumor and the lymph node metastasis. Interestingly, our preliminary data indicate a polyclonal origin of the metastasis. Conclusion: Our protocol is feasible to study comprehensively genetic heterogeneity between primary tumors and metastases on a single-cell level. The single cell amplification products tested for TP53 mutations can be used for several other genetic studies, including genome-wide methods such as CGH. Our initial data revealed an unexpected intratumoral genetic heterogeneity in primaries and metastases of HNSCC.

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 4823. doi:10.1158/1538-7445.AM2011-4823
Abstract 483: CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells
Authors: Taghizadeh Rouzbeth , Noh Minsoo , Huh Yang Hoon , Arosio Beatrice , Sherley James , LaPorta Caterina AM
Published: June 2012

Background- A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main contributes are reported. The first one is the first phenotypic evidence to support the origin of melanoma CSCs from mutated tissue-specific stem cells and the second one is the identification of a more aggressive subpopulation CXCR6+ in melanoma.

Methods/Findings- We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ tumors gave a 2-fold greater mass than tumors from unsorted cells or ABCG2- cells. Furthermore, CXCR6+ cells produced more aggressive tumors. These findings show that CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive melanoma cancer stem cell phenotype than cells selected on the basis of ABCG2, and CXCR6+/ABCG2+ expression.

Conclusions/Significance- The association of more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to preserve self-phenotype, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor progression may provide new targets for cancer prevention and treatment.

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 483. doi:10.1158/1538-7445.AM2011-483
Abstract 4833: A genomic portrait of tumor progression using next-generation sequencing
Authors: Johansson Peter , Wei Jun S. , Chen Qingrong , Bowden Nikola , Badgett Tom C. , Song Young K. , Wen Xinyu , Holman Catherine , Yeh Susan , Khan Javed
Published: June 2012

Genetic alterations are thought to enable cancers to proliferate and survive more effectively or to resist cytotoxic therapies. However, the molecular basis of tumorigenesis and progression is not fully understood. In order to understand the genetic aberrations underlying tumor progression in refractory neuroblastoma, we performed next-generation sequencing on the whole exomes of three sequential tumor samples taken from a high-risk stage 4 neuroblastoma patient at diagnosis, after cytotoxic therapies, and at death, paired with a germline DNA sample from the same patient. Each genomic DNA sample was first partitioned using SureSelect whole human exome kits targeting ∼38Mb of genomic regions, then subjected to sequencing. Each exome sequencing experiment yielded approximately 240 million mappable reads representing 12 billion base pairs. We used DiBayes to identify single nucleotide variants (SNVs). After filtering away the low-quality SNVs, we identified approximately 18,000 high-quality SNVs in each sample, of which ∼7500 are within the protein coding regions. Using the frequencies of the SNVs derived from the sequencing experiments, we discovered a shared LOH signature at the chromosome level among all three tumors indicating they originate from a common cancer progenitor. When we examined the germline DNA, we found about 97% of SNVs for each tumor sample were also found in the germline DNA, and each tumor had more than 300 unique somatic SNVs. These data showed that despite of the shared LOH pattern, tumor genomes are highly unstable at the single nucleotide level. Furthermore, we also found that the three tumor samples shared 64 SNVs which may be potential driver mutations. Twenty of those common 64 SNVs, located on 7 genes, are predicted to be damaging mutations by SIFT algorithm. These mutations are currently being validated using Sanger sequencing, and further RNA-seq in the same tumor samples to investigate for pathway disruption that may lead to chemo-refractory disease.

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 4833. doi:10.1158/1538-7445.AM2011-4833
Abstract 4835: Gonosome-linked expression of PPP2R3B in cutaneous melanoma correlates with distant metastasis free survival
Authors: Spatz Alan , van Kempen Leon C. , Job Bastien , Richon Catherine , Barrois Michiel , Dessen Philipe , Lazar Vladimir , Michiels Stefan , Helbling-Leclerc Anne , Dumay Anne , Petrella Teresa , Batist Gerald , Stas Marguerite , Schadendorf Dirk , van den Oord Joost , Feunteun Jean
Published: June 2012

Background: A strong adverse male effect for melanoma incidence and survival has been reported. No biological explanation, including hormonal differences, has been identified so far. We hypothesize that this gender difference in incidence and survival involves X-linked tumor suppressor genes and/or oncogenes whose expression is affected by X-chromosome alterations.

Methods: DNA and RNA were extracted from 49 fresh frozen primary melanomas (32 females, 17 males, distant metastasis free survival (DMFS) <3 years or follow up >3 years) and subjected to array-based comparative genomic hybridization. Supervised and unsupervised cluster analyses were performed. X-inactivation was studied by Xist RNA in situ hybridization and androgen receptor methylation-sensitive PCR. The expression of a candidate X-linked gene was analyzed by quantitative PCR and correlated with DMFS in uni- and multivariate analyses.

Results: Supervised cluster analyses revealed distinct DNA signatures associated with clinical and histopathological characteristics. Unsupervised clustering identified three groups of which one was composed of predominantly female patients (10/13) and associated with the worst DMFS. This group was characterized by loss of one X-chromosome in 7 of the 13 patients. Loss of X correlated with poor DMFS of female patients (logrank p=0.009). Intriguingly, the lost X chromosome was systematically the inactivated copy. Among the 17 males, Y-chromosome loss correlated with DMFS (logrank p=0.015). We found that the PPP2R3B gene, located within one of the pseudoautosomal regions and thus not inactivated on the inactive X in females, is lost from the Y in males. PPP2R3B encodes a regulatory subunit of protein phosphatase 2 that may negatively control cell proliferation via targets such as CDC6 or RB. Quantitative analysis of PPP2R3B mRNA expression in an independent sample set of melanomas revealed a strong correlation between decreased levels of PPP2R3B expression and poor DMFS irrespective of gender in multivariate analysis (p=0.0007).

Conclusion: Our analyses reveal a specific pattern of X and Y chromosome losses that are associated with melanoma progression. PPP2R3B is a potential gonosome-linked tumor suppressor gene, whose loss of expression correlates with poor DMFS.

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 4835. doi:10.1158/1538-7445.AM2011-4835
Abstract 484: Persistent activation of mesenchymal stromal cells (MSCs) and tissue repair in the etiology of desmoid tumors (DT)
Authors: Carothers Adelaide M. , Rizvi Hira , Heit Yvonne , Hasson Rian M. , Davids Jennifer , Bertagnolli Monica M. , Cho Nancy L.
Published: June 2012

Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from various adult tissues and are implicated in a growing list of sarcomas. Desmoid tumors (DTs) have a clonal origin, are linked with wound healing, and mostly arise in the abdomen of patients with familial adenomatous polyposis (FAP) or sporadically. MSCs home to sites of tissue damage and the persistent activation of these cells in a wound healing context combined with deregulated Wnt signaling may lead to neoplasia. To test this idea, we performed immunohistochemistry on 17 sporadic and FAP-associated DTs from different individuals for the expression of multiple MSC markers (CD73, CD90, and Bmi1). While present in differing numbers, all specimens showed positive cells for all markers, and immunofluorescence (IF) showed co-localization of these markers and CD105 in cells within tumors. Matching adjacent normal fascia from a subset was negative for these antigens. A FAP DT-derived cell line was then made that also co-expressed these MSC markers but not CD34, a negative marker. Fluorescence-activated cell sorting analysis of surface antigen profiles confirmed that >90% of the cell population bore MSC positive, but not negative, markers. Although not genotyped, IF for the C- vs. N-termini of APC confirmed that only truncated APC product was expressed in our DT-derived MSC line. Upon growth for 5-6 weeks in differentiation medium, we confirmed by co-expression of CTGF and Sox9 (obligate chondrocyte markers) plus Alcian blue staining, PPARγ and c/EBPα (obligate adipocyte markers) plus Oil Red staining, and OPN and Runx2 (obligate osteocyte markers) plus von Kossa staining that the DT-derived MSCs were tripotent. In conclusion, this study supports the view that DT etiology involves the persistence of MSCs with deregulated Wnt signaling. The differentiation potential of these cells combined with expression of Bmi1, a transcriptional repressor downstream of Hedgehog and Notch signaling, suggest that inhibitors targeting these pathways may provide a rational systemic therapy to prevent primary or recurrent DT in high-risk 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 484. doi:10.1158/1538-7445.AM2011-484
Abstract 4844: Genomics aberrations and BRCA1 silencing in hereditary triple negative breast tumors in Chilean women
Authors: Tapia Teresa , Sanchez Alejandro , Alvarez Carolina , Cornejo Valeria , Fernandez Wanda , Alvarez Manuel , Camus Mauricio , Carvallo Pilar
Published: June 2012

Breast cancer is one of the most common cancers in women worldwide and the second cause of death by cancer among women in Chile. Either sporadic or hereditary breast cancers show a wide variety of tumor phenotypes, leading to individual clinic responses to specific treatments. Among breast cancer tumors, a group named as triple negative breast cancer (TNBC) does not express hormone receptors and HER2 (ER-, PR-, HER2-). For these tumors no specific treatment complementing chemotherapy has been developed yet. Therefore the study of the genes involved in the progression of TNBC tumor may provide a rationale for the future exploration of therapeutic strategies. TNBC is frequently observed in women carrying a germline mutation in the BRCA1 gene and in approximately 30% of non-carrier women with loss of expression of BRCA1 protein. Our aim, is to identify common genomic aberrations among TNBC tumors and to evaluate BRCA1 silencing by hypermethylation of its promoter in TNBC tumors. It has been described that about 80-90% of TNBC tumors belong to the basal-like molecular subtype, constituting a biological and clinical distinct subgroup. To determine the basal-like subtype we evaluated the expression of Cytokeratin 14 (CK14) marker in 22 hereditary TNBC. Our results revealed that a 63% (14/22) of the tumors express CK14, suggesting that these tumors belong to the basal-like subtype. Additionally, we analyzed the methylation status of the BRCA1 promoter and its correspondent protein expression in the tumors. A 77% (17/22) of the tumors had BRCA1 promoter hypermethylation. Of all tumors 32% (7/22) had absent or reduced nuclear expression of BRCA1. Interestingly, we found 54% (12/22) of the TNBC tumors with a cytoplasmic expression of BRCA1. Finally, we also analyzed through array-CGH, 19 hereditary TNBC tumors to identify common genomic aberrations between these tumors. The array-CGH analysis revealed the gain of the genomic regions 7q34, 15q26.1 and 16p13.3 and the deletion of the regions 1q21.1, 9q33.3, 18q22.1-q22.3 and 19q13.41 in more than 30% of the TNBC tumors. In conclusion, we found that 71% of tumors expressing CK14 have absent or reduced expression of the BRCA1. On the other hand, 61% of tumors with promoter hypermethylation of BRCA1 have loss expression of its protein. Interestingly, the region deleted 1q21.1 was found in 94% of the hereditary TNBC tumors. Financed by FONDECYT 1080595 and CONICYT 24091058.

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 4844. doi:10.1158/1538-7445.AM2011-4844
Abstract 4851: A single-tube protocol for next gen library construction increases complexity and simplifies parallel sample handling
Authors: Park Kyusung , Duan Xiaoping , Meredith Rhonda , Kilzer Jennifer M. , White Eric , Ye Delia , Duncan Cisilya , Sokolsky Tanya , Harris Adam N. , Bennett Rob
Published: June 2012

With the advent of second-generation sequencing technologies, exome- and transcriptome- and whole genome sequencing are tools of choice in studying cancer to detect alterations in the genome correlating with tumorigenesis. It is essential to compare genomic alterations in cancer with matched normal DNA from the same individual largely due to incomplete knowledge of the “normal” variations. There have been concerted efforts for targeted sequencing of cancer-related exomes in a high throughput manner. However, the needs to process multiple samples simultaneously while maintaining the complexity of libraries have not been addressed adequately.

Fragment library generation for single- and paired-end reads is typically performed by a series of enzymatic steps following gDNA shearing by ultrasonication. These may include end-repair, A-tailing, and ligation, each followed by bead- or column-based purification. The clean-ups are necessary to prevent enzyme activity carryover from one step to another.

We have developed a single-tube method to take sheared, bead size-selected DNA through end-repair, A-tailing, and ligation without intermittent purifications. The A-tailing is done by a thermophilic polymerase at a temperature sufficient to denature the end-repair enzymes. Ligation is performed by adding ligase and adaptors after cooling the reaction. A bead- or column-based purification is then performed after the ligation reaction to produce template for amplification.

With the removal of the clean-up steps, the workflow is simplified, allowing faster processing of samples with less hands-on time. We also reduce loss of sample associated with the purifications, increasing the complexity of the output library or enabling a lower sample input concentration. Our method is well suited for labs which are producing multiple libraries each week but have not scaled up to full automation.

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 4851. doi:10.1158/1538-7445.AM2011-4851
Abstract 4857: Improved technology for ribosomal RNA (rRNA) removal from formalin-fixed paraffin-embedded (FFPE) total RNA
Authors: Sooknanan Roy , Radek Agnes , Hitchen John , Khanna Anupama
Published: June 2012

Deep, massively parallel sequencing of cDNA generated from RNA (“RNA-Seq”) is rapidly gaining momentum for transcript profiling, discovery of novel transcripts, and identification of alternative splicing events. Current methods for making sequencer-specific di-tagged DNA fragment libraries for RNA-Seq typically comprise first, preparing rRNA-depleted RNA from total RNA samples that are usually of good quality followed by the synthesis of the di-tagged cDNA sequencing templates. In the past few years however, it has also become evident from microarray and qPCR studies that formalin-fixed paraffin-embedded (FFPE) cancer tissues hold valuable secrets about diseased states. However, RNA-Seq libraries prepared from such highly fragmented FFPE RNA cancer samples yield limited information since these libraries contain a majority of rRNA reads, which decreases sequencing depth and coverage. Current commercially available rRNA-removal kits are not designed to remove fragments of rRNA, which poses a significant limitation for preparing highly informative RNA-Seq libraries from FFPE RNA samples.

Here, we present RNA-Seq results obtained using a novel rRNA removal technology (“Ribo-ZERO™”) and a novel, ligation-free process for preparing directional di-tagged DNA fragment libraries (“ScriptSeq™ Technology”) for RNA-Seq. Using these methods, directional di-tagged DNA fragment libraries can be prepared in about 6 hours from either intact or fragmented (e.g., FFPE) total RNA samples (as little as 100 ng FFPE total RNA sample required). Less than 2 % of the sequence reads from libraries generated from total RNA from either intact or FFPE samples map to rRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNA sequence reads from FFPE RNA samples improves sequence depth and coverage, and increases the percentage of uniquely mapped reads, increasing the information obtained from these diseased samples.

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 4857. doi:10.1158/1538-7445.AM2011-4857
Abstract 4859: Tumor and normal classification of formalin-fixed, paraffin-embedded (FFPE) specimens by transcriptome RNA-seq
Authors: Qu Kunbin , Morlan John , Collin Francois , Millward Carl , Stephans James , Liu Mei-Lan , Jeong Jennie , Baker Joffre , Sinicropi Dominick
Published: June 2012

We have used RNA-seq to profile and compare normal and cancerous human breast tissue. FFPE breast specimens from a total of 24 patients, 12 normal (N) and 12 tumor (T) specimens from surgical resections, were analyzed on an Illumina's GA IIx sequencer. Whole transcriptome RNA-Seq libraries were prepared after depletion of ribosomal RNA by a protocol developed at Genomic Health Inc. (GHI). The analysis was multiplexed across two flow cells using barcoding, with two specimens per sequencing lane (1 T and 1 closely age-matched N library from a different patient). FFPE tissue archive times ranged from 10 to 13 years and they were also closely matched within each lane. To evaluate reproducibility, triplicate libraries were created from 4 of the specimens and analyzed within and across flow cells. Libraries yielded, on average, 19 million 51 bp sequences. R2 values obtained from replicate libraries prepared from the same patient RNA were > 0.9 within and between flow cells. More than 80% of known genes in the human genome were detected in all patients. Several thousand intergenic transcripts were identified by an algorithm developed at GHI. A negative binomial model with tag-wise estimates of dispersion was applied to the known genes and intergenic regions. Inter-patient count variance is generally higher in the set of intergenic sequences than in the set of gene (RefSeq) sequences. Thousands of gene (RefSeq) and intergenic sequences were found to be differentially expressed between T and N tissues. We sought to build classifiers based on flow cell #1 data that could stratify T and N tissues when applied to flow cell #2 data. Sets of genes and intergenic regions were selected for analysis based on high inter-patient count variance. Support vector machine classifiers were trained and then applied to the data from flow cell #2, and also to another GHI tumor/normal RNA-Seq study. Either a set of 100 genes (RefSeq), or a set of 70 intergenic sequences accurately distinguished tumor and normal tissues. Our results offer further evidence of the potential of RNA-Seq for discovery of biomarkers.

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 4859. doi:10.1158/1538-7445.AM2011-4859
Abstract 4869: p27kip1 is a metastasis-associated antigen that promotes cell migration in osteosarcoma
Authors: Li Yiting , Flores Ricardo , Hicks John , Chintagumpala Murali , Lau Ching , Man Tsz Kwong
Published: June 2012

Metastasis is the leading cause of death in osteosarcoma (OS). Despite the advent of cancer therapy, patients with metastasis still have a dismal outcome. Hence, identification and characterization of new cellular targets that are critical to the metastatic process are urgently needed in order to improve both the treatment and detection of metastatic patients. Previous evidence has indicated that cancer patients produce autoantibodies. Many of these tumor-associated autoantibodies target cellular proteins that are essential for tumor formation. In this study, we used a high-density Human Protein Array (HPA) that contains over 8,000 purified proteins, to identify tumor-associated antigens that are associated with metastatic OS at the time of diagnosis. Plasma samples from patients with metastatic (n=10) or localized (n=16) OS, as well as hospitalized children with non-cancerous diseases (n=21) were pooled separately and analyzed using HPA. Totally, 16 autoantibodies were found to be reactive in metastatic OS. Of those, 6 of them showed higher prevalence in the metastatic cases relative to the localized cases and the control cases (p<0.01). One of these autoantibodies targets a cell cycle inhibitor protein, p27kip1, which is involved in tumor suppression and invasion. The higher prevalence of p27kip1 autoantibody in metastatic OS was subsequently validated using a novel HaloLink assay and individual plasma samples. To test if p27kip1 plays a role in metastatic OS, immunohistochemistry was performed on three pairs of metastatic OS cell lines and their parental, non-metastatic cell lines. We found that the p27kip1 protein, which is normally localized in the nucleus, was mislocalized in the cytoplasm in all three metastatic OS cell lines. Functional studies demonstrated that the wild type or CDK- mutant of p27kip1 harboring a nuclear export signal significantly induced OS cell migration in vitro. In summary, our results indicate that mislocalization of p27kip1 can promote metastatic potential of OS. Further characterization of p27kip1 and its related pathways may lead to the development of a novel therapeutic strategy for treating metastatic OS and other sarcomas.

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 4869. doi:10.1158/1538-7445.AM2011-4869
Abstract 4871: Photoreactive stapled peptides to capture cancer proteins and define their sites of interaction
Authors: Braun Craig R. , Mintseris Julian , Gavathiotis Evripidis , Bird Gregory H. , Gygi Steven P. , Walensky Loren D.
Published: June 2012

Protein interactions mediate the pathologic cellular activities that contribute to the development, maintenance, and recurrence of cancer. Whether fleeting or stable, homeostatic or aberrant, protein partnerships and their sites of contact form the basis for discovery of biological pathways, disease mechanisms, and opportunities for therapeutic intervention. When the biological activity of cancer-causing proteins is assigned to discrete structural subdomains, their mode of action, menu of protein targets, and explicit sites of target engagement are frequently unknown. Ideally, the evolutionarily honed substructures could be used to capture and thereby catalogue their protein targets; however, out of context from the whole protein, bioactive subdomains often unfold, resulting in loss of biological shape, potency, and specificity. In an effort to reclaim the enormous capacity of structured peptides to selectively bind and capture their protein targets, we combined peptide alpha-helix stabilization, photoaffinity labeling, proteomic analysis, and structure calculation to generate a new strategy for both (1) expanding our grasp of the static and dynamic alpha-helical interactome of cancer proteins and (2) localizing and targeting alpha-helix/protein interaction sites. We piloted our approach using photoreactive stabilized alpha-helices of BCL-2 domains or pSAHBs and demonstrate that they serve as high fidelity research tools capable of efficiently, selectively, and irreversibly crosslinking both static and dynamic protein targets for proteomic and mechanistic analyses. In addition, the compounds were applied to rapidly and correctly locate protein interaction sites on the anti-apoptotic targets BCL-XL and BCL-w. A unique and compelling feature of this approach is the ability to conduct rigorous validation studies for the identified targets and their sites of interaction. Because stapled peptides can penetrate intact cells and access their intracellular targets, stapled peptide derivatives of the very reagents used for covalent capture can be applied to vet and potentially drug the cancer-causing interactions in vivo.

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 4871. doi:10.1158/1538-7445.AM2011-4871
Abstract 488: Reprogramming cancer cells into terminal differentiation via induced pluripotency
Authors: Matushansky Igor , Zhang Xi
Published: June 2012

Induced pluripotent stem cells (iPSCs) are defined as somatic cells that have been “reprogrammed” (using either the four transcriptional factors Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog, and Lin28) to exhibit embryonic stem cell-like pluripotent differentiation properties. The generation of iPSCs was a great achievement for the field of cell transplantation and tissue engineering. More recently multiple cancer cell lines (e.g., leukemic cells, melanoma cells & gastric cancer cells) have been successfully reprogrammed to the iPSC-state, at least as defined by (1) expression of genes specific to undifferentiated embryonic stem cells; and (2) pluripotency as defined by embryoid body formation in vitro and early markers of commitment to various differentiation lineages.

However, the question that remains unanswered is: can the cancer derived iPSCs complete maturation along a given lineage, be it the lineage from which it was derived (i.e., originally prematurely blocked) or an alternative lineage? If the former were demonstrated it would in essence answer the question of whether global changes in chromatin architecture or ‘epigenetics’ (which are believed to underlie the mechanism of nuclear iPSC reprogramming) can supersede the specific genetic changes (i.e., mutations) in DNA that resulted in the lineage specific differentiation block and subsequent tumorigenesis.

To attempt to answer these questions we generated iPSC-cancer cells from sarcoma cell lines representing undifferentiated sarcomas, osteosarcoma cells, liposarcoma cells, and sarcomas of unknown lineage. While the original sarcoma cell lines could not be differentiated in vitro into mature connective tissue cells, their iPSC-counterparts readily differentiated into multiple mature connective tissue lineages. Furthermore, assessment of proliferation indicated that acquisition of the mature phenotype was concurrent with cessation of proliferation; and thus abrogation of tumorigenicity.

We are currently (1) performing gene expression, miRNA, and global DNA promoter methylation profiling in order to assess the mechanism by which iPSC-based reprogramming of cancer cells over-rides the inherent genetic mutations in these cells and restores the capacity for terminal differentiation; and (2) extending our work to carcinomas.

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 488. doi:10.1158/1538-7445.AM2011-488
Abstract 4886: Investigation of cytoskeletal dynamics in real-time during carcinoma cell migration and invasion in vitro
Authors: Conant Carolyn G. , Schwartz Michael , Ionescu-Zanetti Cristian
Published: June 2012

Understanding the fine details of events leading to the establishment of secondary metastatic foci is complicated by methods that are either endpoint or in vivo. We have devised a unique method to investigate real-time events of cell migration and invasion in vitro. We utilized laminar flow cells with a two-inlet geometry to create compartments of either matrigel or chemotactic stimuli. We then introduced carcinoma cells (HT1080) into opposing neutral compartments and imaged the real-time events associated with cell motility. Use of transient transduction of actin-GFP, the myristolyation/palmitoylation sequence from Lck tyrosine kinase-CFP (MPS) and SV40 nuclear-localization sequence-RFP (BacMam) allowed live investigation of cell motility during chemotaxis and invasion. We found that membrane ruffling occurred within minutes of cell adhesion in the neutral compartment. Chemotaxis and invasion occurred within 4 hours of cell introduction, concurrent with massive and dynamic cytoskeletal reorganization. Migration and cell proliferation continued for the entire duration of the data capture up to 24-hours. This method is further extensible to understanding detailed information regarding signaling, inhibition and fluid dynamics on processes of metastatic cell travel through the vasculature, cell adhesion and extravasation.

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 4886. doi:10.1158/1538-7445.AM2011-4886
Abstract 4893: Microfluidic biochip for the label-free isolation of viablecirculating tumor cells from cancer patients
Authors: Tan Swee Jin , Chen Peng Fei , Lim Wan Teck , Lim Chwee Teck
Published: June 2012

Circulating tumor cells (CTCs) found in the peripheral blood of metastatic cancer patients are rare and this makes them hard to detect. However, the detection and enumeration of CTCs can potentially be used to indicate the severity of the disease or for prognostic and treatment monitoring purposes. Retrieval of viable CTCs can also aid in the study of the phenotypic and genotypic expressions of cancer cells.

We developed an effective label-free detection and isolation of viable CTCs using a microfluidic device that utilizes the unique differences in size and deformability between cancer cells and blood cells. The use of microfluidics also allows the flow conditions to be accurately controlled to achieve an effective and efficient isolation of CTCs. By placing crescent shaped microstructures, termed cell traps, in the path of blood flow in a microchannel, the larger and stiffer CTCs are easily trapped while the blood cells are quickly removed. By reversing the flow, we are able to dislodge and retrieve the trapped CTCs. Using whole blood samples obtained from metastatic lung and kidney cancer patients, we achieved successful detection and separation of CTCs. Here, there is no need for pre-preparation such as staining, tagging or dilution of blood samples as whole blood taken from patient is directly processed in the biochip. The biochip is optically transparent and allows for direct observation using an inverted microscope and for in situ immunofluorescence staining to confirm the presence of CTCs.

Here, we successfully demonstrated the detection and isolation of viable CTCs from cancer patients using the differences in size and deformability of cancer cells and blood cells. This CTC separation process is also label free and will address concerns linked to affinity based isolation methods.

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 4893. doi:10.1158/1538-7445.AM2011-4893
Abstract 4897: A robust 384-well cell migration assay for high content analysis (HCA) of cells treated with anti-cancer therapeutics
Authors: Fronczak Jennifer A. , Hulkower Keren I. , Vogt Andreas
Published: June 2012

Platypus Technologies has developed a first-in-class 384-well cell migration assay to enable high throughput screening (HTS) of anti-cancer compounds and wound healing agents on adherent tumor and endothelial cell lines. The assay is completely automatable with liquid handling systems and high content analysis (HCA) instrumentation. This assay format utilizes a centrally located self-dissolving, non-toxic biocompatible gel (BCG) to form a uniformly sized, cell-free detection zone on tissue culture treated or collagen I coated cell culture surfaces. Cells are seeded into 384-well plates and pattern in an annular monolayer surrounding the BCG. Once the BCG dissolves, cells can migrate into the detection zone previously occupied by the BCG. Dose-dependent inhibition of migration was observed using the actin polymerization inhibitors Cytochalasin D and Latrunculin A, the kinase inhibitors Dasatinib and Sorafenib on HT-1080, and human umbilical vein endothelial cells (HUVECs), and the phosphatase inhibitors NSC95397 and BCI on MDA-MB231 human breast cancer cells. While migration of cells was faster on collagen coated surfaces than on tissue culture treated surfaces, the IC 50 values obtained for the compounds and rank order of potency were similar on both surfaces. This assay format allows an unobstructed view of cell motility throughout the duration of the experiment. Cells may be fixed and treated with multiple stains, including Hoechst or DAPI to visualize nuclei and TRITC-phalloidin to observe F-actin, to enable flexible data capture by either enumerating migrating cells or by calculating the area of closure within the detection zone. The 384-well assay was successfully automated using a Biomek 2000 robotic liquid handler and a Cellomics ArrayScan II high content imaging (HCI) instrument.

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 4897. doi:10.1158/1538-7445.AM2011-4897
Abstract 49: Constructing pathway based predictors of cancer clinical outcome
Authors: Ng Sam , Vaske Charlie , Benz Steve , Durbin James , Szeto Chris , Heiser Laura , Wang Nicholas , Korkola Jim , Bayani Nora , Spellman Paul , Gray Joe W. , Haussler David , Stuart Joshua
Published: June 2012

Current efforts to understand the mechanism of cancer involve using various whole-genome -omics measurements over large patient cohorts. Since a patient response to treatments is highly variable, the challenge then is to integrate the data in order to infer patient-specific disease mechanisms. Recent advances in the analysis of cancer (TCGA ovarian serous carcinoma and glioblastoma multiforme) has shown that a pathway interpretation of DNA copy number, DNA methylation, mRNA expression, and mutations offers a powerful framework for interpreting complex data. The hope is that a pathway-level interpretation of -omics data can identify pathway signatures to predict differences in clinical outcome, whereas traditional machine learning algorithms do not take advantage of the pathway structure of biological data.

We are developing a pathway prediction method based on PARADIGM to discriminate patient outcome based on pathway signatures. Utilizing conditional random fields (CRFs) allow for formal search for a graphical model that optimizes the prediction of a particular variable of interest (VOI) defined by the given classification task, as opposed to a generative model that optimizes the model to explain the data. The method first merges pathways to build a core network around the VOI. The model then seeks to extend the pathway to include new genes and interactions which improve the model's predictive ability on the training data.

Application of our method to 50 breast cancer cell-lines treated with 80 different compounds revealed general and subtype-specific signatures of response in breast cancer. We compared our CRF-based method against a compendium of standard machine-learning algorithms and found that our CRF outperformed all methods on a majority of drugs tested. We also tested the method on a cancer benchmark consisting of a dozen prediction challenges all involving the prediction of clinical outcomes on large patient cohorts using gene expression and copy number data. Again, the CRF model outperformed a majority of classifiers and performed comparably to the best classifiers on most challenges. We expect our method to generalize to a wide variety of biological systems for which high-throughput genomics and functional genomics are available.

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 49. doi:10.1158/1538-7445.AM2011-49
Abstract 490: Cucurbitacin I inhibits tumorigenic ability and enhances radiochemosensitivity in non-small cell lung cancer-derived CD133+ cells
Authors: Chen Kuan-Hsuan , Chou Yueh-Ching , Huang Pin-I , Chen Yi-Wei , Hsu Han-Shui , Chiou Tzeon-Jye , Chiou Shih-Hwa
Published: June 2012

Background: STAT3 signaling is reported to promote tumor malignancy and recurrence in non-small cell lung cancer (NSCLC). The STAT3 pathway has been shown to maintain tumorigenicity and the therapeutic resistance of malignant tumors as well as cancer stem cells (CSC). The aim of this study was to investigate the effect of the strong STAT3 inhibitor, cucurbitacin I, in lung cancer CD133+ cells (NSCLC-CD133+).

Methods: NSCLC-CD133+ and NSCLC-CD133− cells were isolated from 7 NSCLC patients. NSCLC-CD133+ cells treated with or without cucurbitacin I were evaluated for their expression of phosphorylated STAT3 (p-STAT3), tumorigenicity, stemness properties, and resistance to chemotherapeutic drugs and ionizing radiation.

Results: Compared to the parental or NSCLC-CD133− cells, NSCLC-CD133+ cells showed greater tumorigenicity, radioresistance, and high expression of Oct-4, Nanog, and Sox2 at high p-STAT3 levels. Cucurbitacin I treatment at 100 nM effectively abrogated STAT3 activation, tumorigenic capacity, sphere formation ability, radioresistance, and chemoresistance in NSCLC-CD133+ cells. Microarray data suggested that cucurbitacin I inhibited the stemness gene signature of NSCLC-CD133+ cells and facilitated the differentiation of NSCLC-CD133+ cells into NSCLC-CD133- cells. Notably, 150 nM cucurbitacin I effectively blocked STAT3 signaling and downstream survival targets such as Bcl-2 and Bcl-xL expression, and induced apoptosis in NSCLC-CD133+ cells. Finally, xenotransplant experiments revealed that cucurbitacin I plus radiotherapy or chemotherapeutic drugs significantly suppressed tumorigenesis and improved survival in NSCLC-CD133+-transplanted immunocompromised mice.

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 490. doi:10.1158/1538-7445.AM2011-490
Abstract 4903: Identification of damage products derived from C3′-deoxy-3′-thymidinyl radical, a proposed intermediate from DNA-LEEs interactions
Authors: Abdallah Buthina , Trabbic Kevin , Bryant-Friedrich Amanda
Published: June 2012

Radiotherapy uses ionizing radiation (IR) to target deoxyribonucleic acids (DNA) among other biological molecules for cancer treatment. As a result genotoxic, mutagenic and recombinogenic lesions can be formed. Oxidative damage to DNA through IR is partially produced by the secondary particles created along the ionization track. Secondary low-energy electrons (LEEs, 1-20 eV) are the most abundant secondary species produced by primary IR. Although it has been established that LEEs can produce substantial yields of DNA single and double strand breaks, the mechanism of their formation has not been established. Elucidation of the mechanism of damage by LEEs is essential for the development of global models of cellular radiolysis and more efficient methods of cancer radiotherapy. It was proposed that LEE can add to DNA constituents resulting in the formation of transient molecular anion intermediates that eventually lead to bond dissociation. It is believed that carbon-centered radicals on the sugar moiety are a major reactive intermediate formed in this process. One of these is the C3′-deoxy-3′-thymidinyl radical. The synthesis of C3′-deoxy-3′-(selenophenyl)thymidine as a precursor of this radical has been completed. Initial photolysis experiments for photochemical generation of the radical of interest were performed and the formation of the radical intermediate of interest has been established. Identification of damage products derived from this intermediate in nucleosides is currently in progress. The modified nucleoside was incorporated into oligonucleotides using reverse DNA synthesis. Photochemical generation of the C3′-deoxy-3′-thymidinyl radical within DNA was achieved using ultraviolet light (≥ 320nm). Identification of the damage products obtained from the generation of the C3’ radical is currently in progress. Oligonucleotides sequences with different base context were synthesized in order to investigate the effect of sequence on the distribution of damage products. The effect of the concentration of GSH on the damage product distribution is also under investigation. Through these efforts a link will be established between the products of LEEs and the deleterious effects of radiation in cancer radiotherapy.

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 4903. doi:10.1158/1538-7445.AM2011-4903