bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3424: Deciphering the genomic heterogeneity and evolution in malignant melanoma by genomic profiling of clonal tumor populations
Authors: Lorber Thomas , Dietsche Tanja , Perrina Valeria , Barret Michael , Glatz Kathrin , Ruiz Christian , Bubendorf Lukas
Published: May 2015

Background

With the recent improvements in genomic analysis tools, research groups all over the world are intensively deep-sequencing different tumor entities, including malignant melanomas. However, most of these studies are using only a single biopsy per patient, providing only limited insights into genomic tumor evolution. In this study, we performed genomic profiling of sorted clonal tumor populations from several specimens per patient in order to infer genomic heterogeneity in malignant melanomas and genomic evolution during tumor progression.

Methods

A multi-parameter sorting approach was used to flow-sort multiple biopsies from individual melanoma patients. Therefore nuclei were extracted from frozen and FFPE tissues and sorted according to DNA ploidy. SOX10 was used as an additional melanoma parameter to ensure sorting of pure tumor populations. Resulting clonal tumor populations were genomically characterized by usage of high resolution aCGH and deep-sequencing with NGS technologies.

Results

Array CGH and NGS performed on flow-sorted clonal tumor populations (DNA and SOX10) revealed the clonal relationship between multiple tumor biopsies from individual patients. The tumor marker SOX10 allowed us to uncover and sort pure tumor populations, excluding the risk of contribution of non-neoplastic cells in downstream analysis with aCGH and NGS. Taken together the information of ploidy, aCGH and NGS data, we were able to detect chromosome imbalances and allelic frequencies from these populations. Our approach allows us to track the evolution of clonal populations across time and organs and in the context of therapeutic interventions.

Conclusions

Human malignant melanomas are composed of different clonal populations with population-specific genomic aberrations and mutations. The use of SOX10 allowed unraveling of pure tumor populations within the diploid peaks, which would have been obscured by the use of DNA content alone. NGS and aCGH helped to characterize these sorted clonal tumor populations. Further bioinformatic analyses of these sorted clonal tumor populations are fundamental for the understanding of the clonal relationship and genomic heterogeneity and their potential impact on metastasis and therapy response in malignant melanoma.

Citation Format: Thomas Lorber, Tanja Dietsche, Valeria Perrina, Michael Barret, Kathrin Glatz, Christian Ruiz, Lukas Bubendorf. Deciphering the genomic heterogeneity and evolution in malignant melanoma by genomic profiling of clonal tumor populations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3424. doi:10.1158/1538-7445.AM2014-3424
Abstract 3430: Recurrent somatic mutations of nitric oxide synthase NOS3, netrin receptor UNC5C and DNA repair genes in muscle-invasive bladder carcinoma
Authors: Yap Kai Lee , Kiyotani Kazuma , Tamura Kenji , Jang Miran , Montoya Magdeline , Ganshert Cory , Fujioka Tomoaki , Steinberg Gary , O'Donnell Peter , Nakamura Yusuke
Published: May 2015

Bladder cancer is the fourth most common cancer type in males and approximately 72,500 individuals were diagnosed in the United States in 2012. Patients with superficial (low-grade papillary) tumors generally have a good prognosis with a 5-year survival rate exceeding 90%, but those with muscle-invasive or locally advanced disease at surgical resection have a significant risk of recurrence with 5-year survival rates of 30-60%. The systemic chemotherapy options for bladder cancer are very limited, and no new drugs have been approved in the United States for metastatic bladder cancer in over 20 years. Although a number of newer, molecularly targeting drugs have been developed and approved for multiple cancer types in the last two decades, no such drug has been developed for the treatment of bladder cancer. This is likely due, in part, to the relatively limited molecular understanding of invasive bladder cancers and lack of knowledge about potential genetic drivers of invasive forms of the disease. Hence, it is of priority to discover novel genetic pathways involved in the carcinogenesis of muscle-invasive bladder cancer for the assessment of risk stratification and for the development of novel drugs. We analyzed exomes of 43 muscle-invasive bladder carcinomas and identified two novel significantly mutated genes, NOS3 (18.6%) and UNC5C (16.3%), in addition to previously reported frequently mutated genes like TP53 (37.2%) and KDM6A (18.6%). Both NOS3 and UNC5C are known to be involved in the apoptotic pathways, with NOS3 encoding for endothelial nitric oxide synthase (eNOS), and UNC5C for netrin receptor, a member of the dependence receptor family. When we correlated the mutational profiles with clinicopathological outcomes by stratifying patients according to disease recurrence status and observing for specific clustering of genetic mutations, we found that somatic mutations in six DNA repair genes (ATM, ERCC2, FANCD2, RAD51AP1, PALB2, BRCA2) were significantly associated with enhanced recurrence-free survival (hazard ratio of 0.091 with 95% CI of 0.018 to 0.45; P=0.0034) even after adjustment for pathologic TN staging, suggestive of their mutations as favorable prognostic events in bladder cancer.

Citation Format: Kai Lee Yap, Kazuma Kiyotani, Kenji Tamura, Miran Jang, Magdeline Montoya, Cory Ganshert, Tomoaki Fujioka, Gary Steinberg, Peter O'Donnell, Yusuke Nakamura. Recurrent somatic mutations of nitric oxide synthase NOS3, netrin receptor UNC5C and DNA repair genes in muscle-invasive bladder carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3430. doi:10.1158/1538-7445.AM2014-3430
Abstract 3436: Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens
Authors: McClary Andrew C. , Sweeney Robert T. , Biscocho Jewison , Myers Benjamin R. , Neahring Lila , Kwei Kevin A. , Qu Kunbin , Gong Xue , Ng Tony , Jones Carol D. , Varma Sushama , Odegaard Justin I. , Rubin Brian , Troxell Megan L. , Pelham Robert J. , Zehnder James L. , Beachy Philip A. , Pollack Jonathan R. , West Robert B.
Published: May 2015

Rare cancer types are not only understudied, but are typically represented by formalin-fixed paraffin-embedded (FFPE) (rather than freshly-frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non-surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole-transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 21 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly-directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers.

Citation Format: Andrew C. McClary, Robert T. Sweeney, Jewison Biscocho, Benjamin R. Myers, Lila Neahring, Kevin A. Kwei, Kunbin Qu, Xue Gong, Tony Ng, Carol D. Jones, Sushama Varma, Justin I. Odegaard, Brian Rubin, Megan L. Troxell, Robert J. Pelham, James L. Zehnder, Philip A. Beachy, Jonathan R. Pollack, Robert B. West. Ameloblastoma driver mutations revealed by next-generation sequencing of formalin-fixed paraffin-embedded specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2014-3436
Abstract 344: Silencing RBBP6 (retinoblastoma binding protein 6) sensitizes breast cancer cells to staurosporine and camptothecin-induced cell death
Authors: Motadi Lesetja Raymond , Moela Pontsho , Choene Mpho S.
Published: May 2015

Retinoblastoma Binding Protein 6 (RBBP6) is a multi-domain protein that uses its ring finger domain to interact with p53 and pRb tumour suppressor genes. The mechanism by which RBBP6 uses to degrade p53 is still unknown; nonetheless it is well known that RBBP6 promotes cell proliferation in several cancers by negatively regulating p53 via its E3 ubiquitin ligase activity (Ntwasa, 2008). Degradation of p53 by RBBP6 may compromise p53-mediated apoptosis in breast cancer.

This study is intended to investigate, the potential applications of RNA interference (RNAi) to block RBBP6 expression, as well as its subsequent effect on cell growth and apoptosis. To achieve these methodologies the following techniques were used: RT-PCR, western blotting, xCELLigence system and flow cytometry. Our studies indicate that the knockdown of RBBP6 expression by siRNA modulates p53 gene involved in cell death pathways and apoptosis, showing statistically significant gene expression differences. RBBP6 siRNA significantly reduced cell index (CI) compared to the control samples and we observed an inhibition of cellular proliferation in the interval of between 24 and 48 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. These results were further confirmed by flow cytometer which showed some apoptotic activity. About 20.7% increase in apoptosis was observed in cells co-treated with RBBP6 siRNA and camptothecin when compared to camptothecin-only whereas in siRBBP6 and Staurosporine treated there was only 8.8% increase in apoptosis. These findings suggest that silencing RBBP6 may be a novel strategy to promote staurosporine- and camptothecin-induced apoptosis in breast cancer cells.

Citation Format: Lesetja Raymond Motadi, Pontsho Moela, Mpho S. Choene. Silencing RBBP6 (retinoblastoma binding protein 6) sensitizes breast cancer cells to staurosporine and camptothecin-induced cell death. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 344. doi:10.1158/1538-7445.AM2014-344
Abstract 3445: The role of the truncated product of the neuregulin 4 gene in cancer and normal tissues
Authors: Alatsatianos Markella J. , Blackburn Edith , Gullick William
Published: May 2015

The Epidermal Growth Factor Receptor (EGFR) family consists of a group of four transmembrane proteins, EGFR, the Human Epidermal Growth Factor 2 (HER2), HER3 and HER4 that are each involved in cell-signalling. They are activated by ligands, which include the neuregulin (NRG) family of proteins. These proteins are produced as various splice variants from four genes, NRG1-4. Although NRG1 has been extensively investigated, not much is known about the other neuregulins. Thus far, the NRG4 gene has been found to encode five proteins, NRG4 A1, NRG4 A2, NRG4 B1, NRG4 B2 and NRG4 B3. NRG4s B1-3 are truncated structures, which either prematurely terminate as in NRG4s B1 and B2, or have an additional 21 amino acids as in NRG4 B3. Little is known about the structure and function of the NRG4 B3. Previous studies have suggested that it has a different disulphide bond arrangement than that of the other EGF-like ligands. In this study, we have surveyed the expression of NRG4 B3 in breast, lung and colorectal cancers, and found that it is expressed at variable levels. Nuclear magnetic resonance spectroscopy of re-folded NRG4 B3 suggests it has a largely unfolded structure very different from the typical EGF-like molecules. It also has distinct properties in that it is a weak agonist for HER4 signaling and inhibits rather than promotes cell motility in various model systems. Taken together, this suggests that it may have a distinct role in normal and malignant tissues differing in properties to typical EGF-like proteins.

Citation Format: Markella J. Alatsatianos, Edith Blackburn, William Gullick. The role of the truncated product of the neuregulin 4 gene in cancer and normal tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2014-3445
Abstract 3450: Internalization and recycling of RET receptor tyrosine kinase isoforms
Authors: Crupi Mathieu Joseph François , Richardson Douglas S. , Mulligan Lois M.
Published: May 2015

The RET (REarranged in Transfection) receptor tyrosine kinase is crucial for the normal development of the kidneys and enteric nervous system, as well as the regulation of neural-crest cell behaviours, including migration, proliferation, differentiation and survival. RET is also a potent contributor to clinically challenging cancers. Activating mutations in RET can lead to multiple endocrine neoplasia type 2, a cancer syndrome characterized by medullary thyroid carcinoma. Increased activation of wildtype RET receptors has been linked to aggressive breast cancers, and enhanced invasion and perineural spread in pancreatic carcinoma. Two distinct isoforms of the growth factor receptor arise through alternative splicing of the 3’ exons, and are termed RET9 and RET51 to denote the number of amino acids in each C-terminal tail. These isoforms induce unique transphosphorylation patterns on intracellular tyrosine residues, differentially bind downstream signalling proteins, and possess different intrinsic abilities to cause cellular transformation. Here, we explore the differential subcellular localization and trafficking of the two isoforms in human cell line models to better understand RET-mediated cancers. We have observed that RET51 is efficiently matured and localized to the cell membrane, while immature RET9 matures more slowly, leading to lower relative expression on the plasma membrane. Subsequent to RET activation, both isoforms at the cell surface are internalized through AP2-adaptor interactions and the clathrin coated-pit pathway into endosomes. Although RET51 is internalized more rapidly than RET9, a portion of RET51 molecules recycles back to the plasma membrane, maintaining high expression levels at the cell surface. We have shown that RET51 interacts with the sorting protein GGA3, which may allow the long isoform to travel back to the cell surface. Recycling prevents efficient degradation of RET51, allowing for prolonged downstream signaling relative to RET9. Since RET51 has a longer residency time in an endosomal compartment, where sustained Erk1/2 signaling promotes mitogenesis, this may in part explain the greater transforming potential of RET51. Together, our data suggest that differences in RET isoform subcellular localization and trafficking would lead to differences in signal transduction and may in part explain the functional differences observed between RET isoforms.

Citation Format: Mathieu Joseph François Crupi, Douglas S. Richardson, Lois M. Mulligan. Internalization and recycling of RET receptor tyrosine kinase isoforms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3450. doi:10.1158/1538-7445.AM2014-3450
Abstract 3453: Autophagic regulation of the Met receptor tyrosine kinase in breast cancer
Authors: Bell Emily S. , Zuo Dongmei , Park Morag
Published: May 2015

The Met receptor tyrosine kinase (RTK) and its ligand, hepatocyte growth factor (HGF), are potent mediators of epithelial-mesenchymal transition and an invasive growth program. The HGF-Met signaling axis plays a significant role in the development or progression of many human cancers, and elevated Met expression is associated with poor prognosis and the basal subtype in breast cancer. Met signaling is regulated temporally and spatially through endocytic trafficking to degradative or recycling compartments, and delayed Met trafficking to the degradative pathway promotes enhanced cell migration, adhesion-independent growth and tumorigenesis. It is now recognized that autophagy, an intracellular degradative process, utilizes the endocytic system in both formation and maturation of double-membraned autophagosomes. Autophagy can have both tumor promoting and tumor suppressor roles in different contexts or stages of disease. We hypothesized that reprogramming of endocytic membrane system to support an autophagic starvation response may alter Met RTK trafficking, and therefore modulate tumorigenic signaling. To test this, we utilized MDA-MB-231 and BT-549 triple-negative breast cancer cell lines, as well as Hela cells, as models to study the trafficking and signaling of the Met RTK under conditions of autophagic stimulation or inhibition. Following HGF stimulation and receptor internalization, Met engages with core autophagic machinery in a starvation-dependent manner. Increased autophagosome formation decreases Met recycling to the plasma membrane, which can be rescued by shRNA-mediated knockdown of genes required for autophagy initiation. We demonstrate that the autophagic process negatively regulates signaling of Met through the PI3K/Akt and MAPK pathways and decreases Met-dependent cellular responses. These findings identify Met RTK regulation as a potential tumor-suppressive function of autophagy.

Citation Format: Emily S. Bell, Dongmei Zuo, Morag Park. Autophagic regulation of the Met receptor tyrosine kinase in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3453. doi:10.1158/1538-7445.AM2014-3453
Abstract 3457: A possible role for the Hedgehog pathway ligands Desert and Indian in rhabdomyosarcoma
Authors: Roma Josep , Almazán-Moga Anna , Velasco Pablo , Molist Carla , Isaac Vidal , Segura Miguel F. , Soriano Aroa , Luz Jubierre , Laia Paris , Jaume Reventós , Toledo José Sánchez de , Gallego Soledad
Published: May 2015

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children, is thought to derive from embryonic cells committed to developing into skeletal muscle and is divided into 2 major subtypes: embryonal and alveolar (eRMS and aRMS, respectively). Hedgehog pathway-belonging proteins are considered to be key regulators of development in pluricellular organisms, exerting crucial roles in processes such as tissue patterning, proliferation and differentiation. Likewise, Hedgehog (HH) signaling also plays important roles in adult organisms such as stem cell maintenance or tissue repair. The 3 Hedgehog ligands present in mammals are named Sonic (SHH), Indian (IHH) and Desert (DHH). Upon binding of a Hedgehog ligand to Patched, active SMO is detected in the membrane and prevents GLI proteosomal processing. GLI is then translocated to the nucleus where it binds to and regulates GLI-specific promoters. Despite the knowledge gathered to date, the mechanism that drives activation of the pathway in RMS is not well understood; the relative importance of the 3 HH ligands has not been elucidated and a possible role for Desert or Indian HH in the pathogenesis of RMS has not been studied. Previous works reported undetectable levels of SHH in RMS tumors and cell lines with Hedgehog pathway activation, suggesting a mechanism of GLI1-upregulation independent of autocrine SHH expression. Herein, we report extremely low levels of SHH expression in the majority of RMS patients (approximately 70%). Conversely, we report a prominent expression of Desert and Indian HH ligands - instead of SHH - in RMS, thereby supporting the possible existence of an autocrine ligand-dependent activation of the Hedgehog pathway in this neoplasia. Furthermore, the expression of DHH significantly correlates with GLI1 levels, pointing to a crucial role of this ligand for the activation of the pathway in RMS.

Citation Format: Josep Roma, Anna Almazán-Moga, Pablo Velasco, Carla Molist, Vidal Isaac, Miguel F. Segura, Aroa Soriano, Jubierre Luz, Paris Laia, Reventós Jaume, José Sánchez de Toledo, Soledad Gallego. A possible role for the Hedgehog pathway ligands Desert and Indian in rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3457. doi:10.1158/1538-7445.AM2014-3457
Abstract 3461: Profiling the effects of MEK inhibition on kinase phosphorylation and protease secretion using antibody arrays
Authors: Wegner Greta , Eleria Erin , Liao Wen-Chieh , Finkel Dave , James Amy
Published: May 2015

The frequent deregulation of ERK signaling in tumors has led to the development of MEK inhibitors, which have proven to be clinically relevant to malignancies harboring BRAF mutations. Since proliferation and invasion are intertwined aspects of metastasis, antibody arrays were used to measure the effect of multiple MEK inhibitors on 43 kinases and 34 proteases. Treatment of SK-BR-3 cells with MEK inhibitors resulted in decreased ERK (T202/Y204) and STAT3 (S727) phosphorylation. Increased p38 phosphorylation was observed following treatment with multiple inhibitors, but a decrease was observed in response to PD0325901. These differential effects demonstrate the utility of arrays for parsing out crosstalk between signaling pathways. To assess the effects of inhibitors across multiple cancer cell lines, changes in ERK phosphorylation were examined using ELISA. High ERK activation was observed in both RTK-driven and RAF/RAS mutation-containing cells, but did not correlate with sensitivity to MEK inhibition and was instead determined to be inhibitor dependent. Protease profiles were also impacted by MEK inhibition, with reduced MMP secretion detected by antibody arrays and ELISAs in inhibitor treated cells.

Citation Format: Greta Wegner, Erin Eleria, Wen-Chieh Liao, Dave Finkel, Amy James. Profiling the effects of MEK inhibition on kinase phosphorylation and protease secretion using antibody arrays. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3461. doi:10.1158/1538-7445.AM2014-3461
Abstract 3478: Differential level of L-homocysteic acid and lysophosphatidylcholine (16:0) in sera of patients with ovarian cancer
Authors: Kim Yun Hwan , Ju Woong , Kim Seung Cheol , Yoo Byong Chul
Published: May 2015

Ovarian cancer (OVC) is one of the most difficult cancers to detection in the early periods of its development. Therefore there have been numerous attempts to find a biomarker for OVC, but an accurate diagnostic marker is yet to be discovered. In this study, we profiled OVC candidate metabolites from serum to find potential diagnostic marker for OVC. Information of low-mass-ions (LMIs) in serum was obtained by using MALDI-TOF. MALDI-MS analysis of each serum sample was repeated six times in order to reduce experimental errors. Intensity of LMI mass peak was normalized using total peak area sums. Normalized intensity of LMI was used in principal component analysis-discriminant analysis (PCA-DA) to discriminate 142 OVC from 100 controls. LC-MS/MS was used to identify the selected LMIs. Extracted ion chromatogram (EIC) analysis was used to measure the relative quantity of candidate metabolite from the LMI mass peak areas. The concentration of common metabolite in serum was determined by ELISA. Top 20 LMI mass peaks with weigh factor over 0.05 were selected to separate OVC from controls. Among them two LMIs with 184.05 m/z and 496.30 m/z were identified as L-homocysteic acid (HCA) and lysophosphatidylcholine (LPC) (16:0), respectively. Relative quantity of LPC (16:0) was decreased in OVC serum (P=0.05), whereas the amount of HCA was significantly higher in OVC serum (P<0.001). HCA was not detected in 59 cases out of 63 controls, but most cases of OVC (16 out of 25) showed significantly higher amount of HCA. When the cutoff was 10 nmol/ml, sensitivity and specificity of HCA were 64.0% and 96.9%, respectively. Level of LPC (16:0) was significantly correlated with tumor grade (P=0.045). Both HCA and LPC (16:0) showed correlation with stage and tumor histology, but limited sample number could not assign the statistical significance. HCA alone showed a potential power of biomarker for OVC. Even the stratified screening including LPC (16:0) did not significantly increase the power for OVC screening, our present study showed that a good example of profiling of LMIs in serum can be useful to select and identify candidate metabolites for OVC screening.

Citation Format: Yun Hwan Kim, Woong Ju, Seung Cheol Kim, Byong Chul Yoo. Differential level of L-homocysteic acid and lysophosphatidylcholine (16:0) in sera of patients with ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3478. doi:10.1158/1538-7445.AM2014-3478
Abstract 348: JMJD3 promotes the survival of diffuse large B-cell lymphoma subtypes via distinct mechanisms
Authors: Zhang Yan
Published: May 2015

Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin's lymphoma, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL. Dysreguation of genes related with B cell differentiation contributes to the tumorigenesis of DLBCL; the. Jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase, plays a known role in antigen-driven B-cell differentiation in GCB cells. But the mechanism of JMJD3 in DLBCL progression remains poorly uncerstood. Here we show that JMJD3 exerts an anti-apoptotic effect in both ABC and GCB subtypes of DLBCL. The downregulation of JMJD3 increased apoptosis among DLBCL cells. In the ABC subtype, JMJD3 regulates the interferon regulatory factor 4 (IRF4) in a demethylase activity-dependent fashion. Interestingly, IRF4 could reciprocally regulate the expression of JMJD3, forming a positive feedback regulation loop. The expression of IRF4 was sufficient to rescue survival induced by the JMJD3 suppression selectively in the ABC type. In the GCB subtype, JMJD3-mediated survival was dependent upon increased expression of the anti-apoptotic protein BCL2. Treatment with JMJD3 siRNA could also reduce tumor volume concordant with increased apoptosis of DLBCL cells. Our results demonstrate a previously unrecognized role of JMJD3 in the survival of both the ABC and GCB subtype of DLBCL and provide molecular insight into the mechanisms by which the JMJD3-IRF4 or JMJD3-BCL2 axis controls this process.

Key words:

DLBCL, apoptosis, JMJD3, IRF4, BCL2

Citation Format: Yan Zhang. JMJD3 promotes the survival of diffuse large B-cell lymphoma subtypes via distinct mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 348. doi:10.1158/1538-7445.AM2014-348
Abstract 3489: A novel investigation into chemotherapy-induced peripheral neuropathy using iPSC-derived human neurons
Authors: Komatsu Masaaki , Wheeler Heather E. , Wing Claudia , Delaney Shannon , Dolan M Eileen
Published: May 2015

Chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting side effect of many anti-cancer drugs including taxanes, vinca alkaloids and platinating agents. These drugs represent standard of care for common cancers, however there are no effective treatments and no means to identify patients at risk for CIPN. The mechanisms underlying CIPN have not been precisely determined and few human neuronal models to study CIPN exist. Recent technology has allowed for the differentiation of induced pluripotent stem cells (iPSC) to human neurons. We have utilized genetically diverse human neurons as a model to study the effects on chemotherapy on neuronal health and on neurite morphology including neurite outgrowth, processes and branching. Upon treatment of human neurons with paclitaxel, vincristine or cisplatin (1 nM to 100 µM) for 72 hours, we identified reproducible decreases in cell median neurite process length per magnitude of drug concentration of 12-14%, 6-18% and 2-4%, respectively. No decrease in neurite length was observed with hydroxyurea, an agent that does not cause neuropathy. The morphological changes differed across drugs, with vincristine treatment showing the most dramatic effect on neurite outgrowth, paclitaxel showing an intermediate effect, and cisplatin showing no effect up to 1 µM of drug. To determine whether the model could be used for evaluation of genes important in CIPN, we used siRNA to decrease expression of TUBB2A, a gene known to be associated with paclitaxel-induced neuropathy. Transient knockdown of TUBB2A caused decreases of mean neurite process length (interaction P = 2.2 x 10-8) and total neurite outgrowth (interaction P = 1.8 x 10-10) of neurons 48 hours post-paclitaxel treatment. We are using this novel approach to screen agents that may be used to prevent or treat CIPN.

Citation Format: Masaaki Komatsu, Heather E. Wheeler, Claudia Wing, Shannon Delaney, M Eileen Dolan. A novel investigation into chemotherapy-induced peripheral neuropathy using iPSC-derived human neurons. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3489. doi:10.1158/1538-7445.AM2014-3489
Abstract 3498: A systematic approach to evaluate and select preclinical study workflow software applications for oncology
Authors: Ibsen Eric M. , Kumer Jeffrey
Published: May 2015

The use of software applications to automate the workflow specific to preclinical oncology trials can significantly improve labor efficiency, data quality & security while preserving both methodology and results in a searchable database in perpetuity. The typically daunting and complex selection process can be simplified, facilitated and expedited by applying a systematic approach derived from those developed and used by oncology departments in some large pharmaceutical companies. Criteria common across these companies have been identified and combined into a single codified evaluation method. The first step is to identify and define a detailed set of system requirements based on the needs for each user level: technicians, scientists, statisticians, analytical labs, management and other stakeholders involved with animal studies. Some requirements are specific to specific animal models and must be considered for inclusion. Requirements may include functions such as staff intercommunications, protocol development, ACUC restrictions, tumor induction methods, treatments, group composition, tasks, measurements, schedules, analysis, graphing and reporting methods, etc. Each requirement is then assigned a weight based on its’ relative importance to the users. Frequency of use is also taken into consideration when developing a requirement weight. Software applications under consideration are evaluated by scoring the extent to which each requirement is met. To determine the top applications for in-house evaluation on actual studies, a preliminary evaluation of Requirement Scores may be performed by referencing user manuals. Requirement Scores are multiplied by their predetermined weight to derive a Net Requirement Score (NRS). NRSs are summed to achieve a Total Score. The applications selected with the highest NRS are usually evaluated in the lab on actual studies. Requirements are then scored by a small team of users in regular lab use. The Total Score is used to identify which software best meets the needs of the research organization as a whole. The implementation of a systematic approach to evaluate software for animal study workflow automation can save time and help ensure the objective selection of the best system available for a lab.

Citation Format: Eric M. Ibsen, Jeffrey Kumer. A systematic approach to evaluate and select preclinical study workflow software applications for oncology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3498. doi:10.1158/1538-7445.AM2014-3498
Abstract 3503: Somatic mutations and aberrant expression of RNF43 are recurrently found in intraductal papillary mucinous neoplasms of the pancreas
Authors: Sakamoto Hitomi , Kuboki Yuko , Hatori Takashi , Yamamoto Masakazu , Sugiyama Masanori , Shibata Noriyuki , Shimizu Kyoko , Shiratori Keiko , Furukawa Toru
Published: May 2015

Background: RNF43 encodes ring finger protein 43, an ubiquitin ligase, and is recently uncovered as mutated in intraductal papillary mucinous neoplasms of the pancreas (IPMNs). We studied mutations and aberrant expressions of RNF43 and their associations with molecular and clinicopathological features in IPMNs. Materials and methods: Studied were 178 IPMNs operated upon between 2001 and 2010 at Tokyo Women's Medical University Hospital. Formalin-fixed and paraffin-embedded tissues of the 178 cases and frozen tisssues of 56 of the cases were subjected for immunohistochemical and the next generation semiconducter sequencing analyses, respectively. Detected mutations were validated by Sanger sequencing. Results: Expression of RNF43 was reduced in 60 (33.7%) of the 178 IPMNs. Somatic mutations in RNF43 were found in 10 (17.9%) of the 56 examined cases, in which frameshift mutations, a nonsense mutation, and missense mutations were found in 5, 1, and 4 cases, respectively. These mutations were significantly associated with RNF43 expression (p = 0.008) and GNAS mutation (p = 0.012), however, not associated with tumor grade, histological subtype or prognosis. Conclusion: These results indicate that RNF43 may function as a tumor suppressor and play a role in development of IPMN.

Citation Format: Hitomi Sakamoto, Yuko Kuboki, Takashi Hatori, Masakazu Yamamoto, Masanori Sugiyama, Noriyuki Shibata, Kyoko Shimizu, Keiko Shiratori, Toru Furukawa. Somatic mutations and aberrant expression of RNF43 are recurrently found in intraductal papillary mucinous neoplasms of the pancreas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3503. doi:10.1158/1538-7445.AM2014-3503
Abstract 3504: Regulation of cell cycle progression by Ikaros in leukemia
Authors: Dovat Elanora , Payne Jonathon , Casiano Carlos M. , Sloane Justin , Gowda Chandrika , Payne Kimberly J. , Dovat Sinisa , Song Chunhua
Published: May 2015

Control of cell cycle progression is achieved by the coordinated function of a large set of genes that are highly conserved in eukaryotic organisms. Malignant cells have impaired regulation of cell cycle progression which results in uncontrolled cellular proliferation. Thus, understanding the regulation of cell cycle progression in malignant cells is essential to advance our knowledge of the process of malignant transformation and for designing novel treatments. Ikaros is a zinc finger protein that acts as a tumor suppressor in leukemia. The loss of Ikaros activity due to deletion or mutation has been associated with the development of high-risk B-cell acute lymphoblastic leukemia (B-ALL), as well as with T-cell ALL and acute myelogenous leukemia (AML). Ikaros binds DNA and regulates transcription of its target genes via chromatin remodeling. The mechanism of Ikaros tumor suppressor activity is largely unknown. Here, we present evidence that Ikaros regulates cell cycle progression in leukemia. Using quantitative Chromatin Immunoprecipitation assay (qChIP), we demonstrate that Ikaros binds in vivo to promoter regions of several genes that regulate cell cycle progression in B-ALL cell lines and in primary cells from patients with B-ALL. To study how Ikaros regulates transcription of these genes, luciferase reporter assays were performed. The promoter regions of three Ikaros target genes were cloned into luciferase reporter constructs. Each of these constructs has been co-transfected with Ikaros or an empty vector (as a negative control) into HEK 293T cells. Results showed that Ikaros represses transcription of the three genes that promote cell cycle progression. Overexpression of Ikaros in leukemia cells by retroviral transduction results in reduced transcription of the cell cycle promoting genes, as evidenced by quantitative real-time PCR (qRT-PCR), as well as cell cycle arrest. These data suggest that Ikaros regulates cell cycle progression in leukemia by direct repression of the transcription of the genes that promote cell cycle progression, and identifies one mechanism of Ikaros function as a tumor suppressor in leukemia.

Citation Format: Elanora Dovat, Jonathon Payne, Carlos M. Casiano, Justin Sloane, Chandrika Gowda, Kimberly J. Payne, Sinisa Dovat, Chunhua Song. Regulation of cell cycle progression by Ikaros in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2014-3504
Abstract 3505: Regulation of Cyclin A1 promoter by the Wilms tumor gene, WT1
Authors: Pandey Sony , Moazam Mustafa , Kuerbitz Steven J. , Fraizer Gail C.
Published: May 2015

Cyclin A1(CCNA1) is a unique type of Cyclin A expressed predominantly in highly proliferative tissues such as the testis and bone marrow. In hematopoietic cells CCNA1 expression has been observed in CD34+ progenitor cells, leukemia stem cells and leukemia cell lines. Similarly, Wilms tumor 1 (WT1) gene expression has been observed in these highly proliferative, undifferentiated cells. Regulation of CCNA1 expression is poorly understood, but the CCNA1 gene promoter is a highly GC-rich TATA-less sequence, containing three potential WT1 binding sites. Chromatin immunoprecipitation analysis of two of the three sites revealed binding of the WT1 protein to chromatin in K562 leukemia cells. To assess the role of WT1 in CCNA1 regulation, activation of the CCNA1 promoter by WT1 was measured using luciferase reporter constructs in K562 cells. The distal construct (-1180/+145 ) contains two potential WT1 sites, and the activity of this reporter construct was up-regulated by WT1 co-transfection. In contrast, the proximal (-454/+145) promoter region contains no canonical WT1 binding sites. Suprisingly, the proximal promoter was also up-regulated by WT1 co-transfection. Transcription factor binding site analysis revealed 6 GC boxes -within the core promoter region of the proximal promoter construct that we predicted could serve as non-canonical WT1 binding sites. To determine the importance of these GC boxes for WT1 binding, we treated K562 cells with Mithramycin A, which blocks binding at GC rich sequences. We found that Mithramycin A treatment decreased the activity of both the distal and proximal promoter constructs. Finally we demonstrated that WT1 mediated activation of CCNA1 promoter required an intact DNA binding domain, as a mutant WT1 expression construct lacking the zinc finger domain failed to activate any of the CCNA1 promoter constructs. Overall these results show that WT1 binding can up-regulate the CCNA1 promoter. This observation supports our previous finding that overexpressing WT1 in K562 cells increased CCNA1 mRNA. These data suggest that elevated WT1 expression could mediate increased CCNA1 expression in leukemia cells and thereby enhance proliferation. Overall, our results are consistent with the hypothesis that both WT1 and CCNA1 behave as oncogenes in leukemia.

Citation Format: Sony Pandey, Mustafa Moazam, Steven J. Kuerbitz, Gail C. Fraizer. Regulation of Cyclin A1 promoter by the Wilms tumor gene, WT1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3505. doi:10.1158/1538-7445.AM2014-3505
Abstract 3506: Heregulin induces transformation of mammary epithelial cells via HER3 activation
Authors: Lupu Ruth , Khurana Ashwani , Lugo Ghiara , Guerrico Anatilde Gonzalez
Published: May 2015

Heregulin (HRG)s are high-affinity combinatorial ligands for the HER family of tyrosine kinase receptors. HRG induces heterodimerization and cross-talk between members of the HER tyrosine kinase receptor family. HRG interaction with HER3 induces heterodimerization between HER3 and other HER receptors, including epidermal growth factor receptor (EGFR) and HER2, resulting in cross-talk-induced tyrosine phosphorylation. Several studies have revealed that HRG is expressed in about 40% of invasive breast carcinomas, many of which are triple negative breast carcinomas. We have previously demonstrated that HRG expression induces tumorigenesis and metastasis of MCF-7 cells. We tested the effect of HRG on non-committed MCF10A cells. MCF-10A cell line is an immortalized, non-transformed epithelial cell line derived from human fibrocystic mammary tissue. These cells lack the ability to form tumors in nude mice and the ability to grow in anchorage-independent assays. We found that forced expression of HRG in MCF10A induced cellular transformation, while the control MCF10A/Vector cells did not growth. MCF10A/HRG cells express constitutive phosphorylation of HER3 and induction of the signaling cascade. HER3 phosphorylation occurs as a result of HRG induction of HER3 heterodimerization with EGFR in an autocrine manner. Lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2 and EGFR pathways, blocked HER3 phosphorylation and the resulted signaling in MCF10A/HRG cells and prevented their anchorage-independent growth demonstrating that HER3 phosphorylation is responsible, at least in part, for the transformation of MCF10A/HRG cells. This study provides an alternate targeting prospective for breast carcinomas that express HER3 and HRG by targeting the autocrine-loop (HRG/HER3) and thus preventing HRG from binding to HER3, disrupting the possible receptor heterodimerization, and leading to blockage of the proliferative signals and promoting inhibition of growth.

Citation Format: Ruth Lupu, Ashwani Khurana, Ghiara Lugo, Anatilde Gonzalez Guerrico. Heregulin induces transformation of mammary epithelial cells via HER3 activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3506. doi:10.1158/1538-7445.AM2014-3506
Abstract 351: Comparative studies on SIRT6 and SIRT7 interactomes: Implications of important roles in cancer via associating with interacting partners
Authors: Lee Namgyu , Kwon Jung-Hee , Park Sung Jin , Choi Kwan Yong
Published: May 2015

Sirtuins are NAD+-dependent deacetylases that regulate a range of cellular processes. Although anti-aging effects of sirtuins have been proposed, the proteins interacting with SIRT6 and SIRT7 remain elusive. In the current study, we identified SIRT6- and SIRT7-interacting proteins and compared their interactomes. Our data revealed 129 novel interacting proteins for SIRT6 and 122 for SIRT7 while confirming 7 and 111 previously identified proteins for SIRT6 and SIRT7, respectively. Comparison of the SIRT6 and SIRT7 interactomes under a same experimental condition disclosed 111 shared proteins indicated that functional associations. The functional networks of interactomes exhibited that the binding partners are associated with important biological processes in aging and age-related diseases including cancer. Two proteins, NPM1 and NCL localized in the nucleus and interacted with SIRT6 and SIRT7. NPM1 was identified as a novel substrate and its acetylation level decreased by both SIRT6 and SIRT7. Additionally, in senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased level of SIRT6 and SIRT7, suggesting that acetylation of NPM1 is regulated by SIRT6 and SIRT7 in the aging process. Our findings provide insights on functional relationship between SIRT6 and SIRT7 as well as their roles in DNA repair, chromatin assembly and cancer cell proliferation, and aging.

Citation Format: Namgyu Lee, Jung-Hee Kwon, Sung Jin Park, Kwan Yong Choi. Comparative studies on SIRT6 and SIRT7 interactomes: Implications of important roles in cancer via associating with interacting partners. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 351. doi:10.1158/1538-7445.AM2014-351