bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B26: Melanoma cells induce stem cells like fenotype of normal human keratinocytes
Authors: Kodet Ondrej , Lacina Lukas , Dvorankova Barbora , Kolar Michal , Strnad Hynek , Smetana Karel
Published: August 2014

Introduction: Nodular melanoma is one of the most life threatening tumors with frequently poor therapeutic outcome. Similarly to other the tumors, the intercellular interactions may be of crucial importance for biological properties of melanoma and course of the disease. This study is mapping the effect of melanoma cells and melanoma associated fibroblasts on keratinocytes as compared with effect of neural crest stem cells.

Methods: 100 samples removed during radical melanoma resection were analyzed by histology and 20 of them by immunohistochemistry and compared with in vitro models. For in vitro models of intercellular interactions we co-cultured in transwell systems melanoma cells or neonatal melanocytes or neural crest stem cells respectively with human normal keratinocytes. Further, we employed also melanoma associated fibroblasts and normal human dermal fibroblasts respectively in above mentioned culture system. Transcriptional analysis of these models was carried out in collaboration with the Institute of Molecular Genetics of the ASCR on the whole genome level by using Illumina microarrays.

Results: In tissue surrounding nodular melanoma we have observed significant hyperplasia of epithelial layer with aberrant suprabasal expression of keratin 14 (K14) along with absence of K10 - marker of differentiated epidermal keratinocytes. In vitro models revealed that melanoma cells, neural crest stem cells and also melanoma associated fibroblasts were able to influence expression of K14, but also expression of K19 and K8 as well as of vimentin in cocultured keratinocytes. Cytokines and growth factors such as FGF2, CXCL1, IL8 and VEGFA seem to participate in this activity of melanoma cell to keratinocytes.

Conclusion: Melanoma cells as well as melanoma associated fibroblasts are able to modify the landscape of tissue surrounding the proliferating malignant clone. These interactions result in maintenance of low differentiation status of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma, but the functional significance for melanoma biology requires further analysis.

This study was supported by the Charles University project PRVOUK 27-1 and by the Grant Agency of the Czech Republic project no 304/121/333.

Citation Format: Ondrej Kodet, Lukas Lacina, Barbora Dvorankova, Michal Kolar, Hynek Strnad, Karel Smetana, Jr.. Melanoma cells induce stem cells like fenotype of normal human keratinocytes. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B26.
Abstract B29: Defining subclonal signaling heterogeneity in glioblastoma multiforme (GBM)
Authors: Lan Xiaoyang , Meyer Mona , Reimand Jüri , Zhu Xueming , Kushida Michelle , Head Renee , Clarke Ian , Bader Gary , Dirks Peter
Published: August 2014

Glioblastoma Multiforme (GBM, WHO grade IV) is the most common and malignant brain tumor of the adult central nervous system, with a median survival of between 12 and 15 months. Interestingly, the presence of genetically unique subclones in GBMs has been previously implicated in GBMs using Fluorescence in situ hybridization (FISH) and surgical multisampling approaches.1,2 The purpose of this study are to unequivocally demonstrate clinically relevant phenotypic heterogeneity in GBM subclones, and to identify genetic and phenotypic biomarkers predictive of subclonal drug responses. Previous work in the Dirks laboratory has allowed for the generation of 44 single-cell derived clonal cultures from 4 GBM tumors, by Fluorescence Activated Cell Sorting (FACS) and clonal expansion in vitro. Comparisons of clones derived from the same tumor uniquely allows for the identification and characterization of genetic and functional intratumoral diversity in GBM. We demonstrate that single-cell derived clonal cultures from the same primary tumor can differ in key phenotypes such as proliferation in vitro and responsiveness to the chemotherapeutic Temozolomide. Our work has also shown that expression of the oncogene EGFRvIII is exclusive to some clones within a tumor, and not others. Profiling of basal and activated EGFR signaling states also revealed subclonal variability in pERK and pS6 activity, suggesting subclonal differences in pathway dependencies which could be exploited for targeted therapies. Importantly, we observed clonal differences in sensitivity to the EGFR small molecule inhibitor Erlotinib, suggesting that clonal phenotypes can be correlated to differences in signaling activity between tumor subclones. The existence of inherently drug resistant clones in GBM tumors may lead to ineffective therapies and tumor relapses, and subclonal interrogation of patient specimens may lead to better treatment stratification.

References

1) Snuderl, M., L. Fazlollahi, et al. “Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma.” Cancer Cell 20, 810-817(2011).

2) Sottoriva, A., I. Spiteri, et al. (2013). “Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics.” Proc Natl Acad Sci USA 110, 4009-4014(2013).

Citation Format: Xiaoyang Lan, Mona Meyer, Jüri Reimand, Xueming Zhu, Michelle Kushida, Renee Head, Ian Clarke, Gary Bader, Peter Dirks. Defining subclonal signaling heterogeneity in glioblastoma multiforme (GBM). [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B29.
Abstract B30: Identification of new regulator in p53-mediated cellular senescence
Authors: Shin Jae-Sik , Hong Seung-Woo , Lee Dae-Hee , Moon Jai-Hee , Kim Jeong Eun , Hong Yong Sang , Kim Kyu-pyo , Lee Jae-Lyun , Choi Eun Kyung , Lee Jung Shin , Jin Dong-Hoon , Kim Tae Won
Published: August 2014

The downstream events and target genes of p53 in senescence responses are not fully understood. Here, we identify a novel regulator, A-core gene, in p53-mediated cellular senescence. Overexpression of A-core in p53-deficient mouse embryonic fibroblasts (MEFs) accelerates senescence, whereas A-core gene knockdown by small interfering RNA (siRNA) leads to escape from p53-mediated senescence in p53-expressing MEFs. A-core overexpression also increased intracellular levels of reactive oxygen species (ROS). The ROS inhibitor N-acetyl-L-cysteine rescued A-core overexpression-induced senescence. Surprisingly, the elevated levels of ROS that accompanied A-core overexpression were paralleled by an increase in p21 expression. siRNA-mediated knockdown of p21 in A-core overexpressing MEFs abrogated the A-core-dependent increase in ROS levels, indicating that A-core acts through p21 induction and subsequent ROS elevation to trigger senescence. In addition, expression of the cell proliferation-related proteins cyclin E and B was decreased after A-core expression. Collectively, these results suggest that A-core is a downstream target of p53 that is sufficient to induce p21 expression and ROS production, and is necessary for p53-mediated senescence.

Citation Format: Jae-Sik Shin, Seung-Woo Hong, Dae-Hee Lee, Jai-Hee Moon, Jeong Eun Kim, Yong Sang Hong, Kyu-pyo Kim, Jae-Lyun Lee, Eun Kyung Choi, Jung Shin Lee, Dong-Hoon Jin, Tae Won Kim. Identification of new regulator in p53-mediated cellular senescence. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B30.
Abstract B31: Cell-based screening of novel anticancer molecules for prostate cancer
Authors: Santos Karen Cristina dos , Malavolta Isadora , Leitao Andrei
Published: August 2014

Metastatic and mutant androgen receptor prostate cancer are still of major concern for chemotherapy, lacking an efficacious approach to tackle this life threatening disease. This is so, because of the resilience to androgen ablation therapy. To accomplish with that, new molecules for prostate cancer have been tested in different in vitro models by academic groups and pharmaceutical entrepreneurs. In this work, novel molecules were tested in cell line that do not express androgen receptor (DU145) or cells harboring the mutant androgen receptor (LNCaP). Mouse fibroblast (Balb-c 3T3) cells were also tested in this cell-based screening to check for selectivity. These novel molecules came from previous studies using computational medicinal chemistry tools regarding mutant androgen receptor, surviving and mTOR inhibitors. All biological results were compared with the reference molecules bicalutamide, rapamycin, doxorubicin and YM155. The first screening comprised MTT colorimetric assay, further confirmed by flow cytometry studies, including cell cycle analysis and apoptosis indicators. Several novel molecules presented activity according to the screenings using prostate cancer cell lines. YM155 (a survivin inhibitor) was the only active molecule from the reference set when using the DU145 cells. Besides that, YM155 was the most selective molecule from the whole compound series (composed by reference and test sets). These phenotypic screenings provided the first evidence of anticancer molecules amenable for further chemical optimization.

Citation Format: Karen Cristina dos Santos, Isadora Malavolta, Andrei Leitao. Cell-based screening of novel anticancer molecules for prostate cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B31.
Abstract B41: EMC1 influences cellular calcium homeostasis and promotes breast cancer cell migration and proliferation
Authors: Molina Roberto Augusto Silva , Silva Rodrigo Ribeiro , Costa Roberta Ribeiro , Banzi Silmara Reis , Espreafico Enilza Maria
Published: August 2014

Endoplasmic Reticulum Membrane Complex protein 1 (EMC1), also named KIAA0090, was recently identified as part of a complex with six proteins (EMC1-EMC6) proposed to participate in the folding/maturation of membrane proteins, but the mechanisms remains unclear. High throughput studies reveal that EMC1 gene is up-regulated in many types of cancer and up-regulation correlates to poor survival rates. It has been known that there is a close relationship between proteins involved in the unfolded protein response (UPR) and tumor maintenance. Here, we investigated the involvement of EMC1 in breast cancer cell proliferation and migration. We showed by qPCR that EMC1 gene is overexpressed in breast cancer cell lines in comparison to immortalized epithelial breast cells. Next, we stably overexpressed EMC1 in MCF-7 and SKBR-3 cell lines. Overexpression led to an increase in proliferation rates, clonogenic capacity, and migration in wound-healing assays. Consistently, EMC1 knockdown in SKBR-3 cells led to a decrease of cell migration and clonogenic capacity. Furthermore, we assessed by Fluo3 the involvement of EMC1 in the regulation of intracellular calcium levels in SKBR-3 cells in response to thapsigargin treatment, which inhibits the endoplasmic reticulum Ca2+ ATPase. SKBR-3 control cells showed an expected increment in cytosolic calcium levels following thapsigargin addition. Interestingly, cytosolic calcium increments were about 50% higher in EMC1 overexpressing cells and nearly 10-fold lower in knockdown cells, suggesting a role for EMC1 in the control of cellular calcium homeostasis. EMC1 could function in the maturation/assembly of calcium channels or calcium pumps, thereby promoting breast cancer cell migration and maintenance.

Citation Format: Roberto Augusto Silva Molina, Rodrigo Ribeiro Silva, Roberta Ribeiro Costa, Silmara Reis Banzi, Enilza Maria Espreafico. EMC1 influences cellular calcium homeostasis and promotes breast cancer cell migration and proliferation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B41.
Abstract B44: A role for endothelial αvβ3 integrin in breast tumor cell migration
Authors: Montenegro Cyntia Freitas , Durante Araceli Cristina , Micocci Kelli Cristina , Pereira Antonio Carlos Manucci , Selistre-de-Araujo Heloisa Sobreiro
Published: August 2014

Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood and evasive resistance of antiangiogenic therapy has been reported. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin mediated downstream signaling cascades and cell migration. Recently, we reported that DisBa-01 strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. Here, DisBa-01 was submitted to adhesion under flow and transmigration assays, both using different DisBa-01 concentrations and β3-siMDA-MB-231 cells. In the first assay, labeled tumor cells incubated with Disba-01 perfused for 5 minutes through a chamber containing endothelial cells as coat. In the second, labeled tumor cells incubated with DisBa-01 were attracted by complete medium and transmigrated through an endothelial cell coat. Individually, assays were photographed and the cells quantified. Results show that DisBa-01 dose-dependently inhibited adhesion of MDA-MB-231 cells to human endothelial cells under flow conditions, similarly to the effect observed by β3-siMDA-MB-231 cells. In addition, the disintegrin or β3-silencing inhibited the transmigration of tumor cells through endothelial cells in vitro. These results demonstrate that endothelial αvβ3 integrin has a key role in tumor cell adhesion and transmigration through the endothelium under flow conditions.

Support: Fapesp (grant no. 1998/14138-2), CNPq, and CAPES (Brazil)

Citation Format: Cyntia Freitas Montenegro, Araceli Cristina Durante, Kelli Cristina Micocci, Antonio Carlos Manucci Pereira, Jr., Heloisa Sobreiro Selistre-de-Araujo. A role for endothelial αvβ3 integrin in breast tumor cell migration. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B44.
Abstract B45: A cell-based screening to identify nucleolar disruptors in cancer cells
Authors: Morgado-Palacin Lucia , Llanos Susana , Blanco-Aparicio Carmen , Megias Diego , Pastor Joaquin , Serrano Manuel
Published: August 2014

Cancer cells require highly elevated levels of ribosome biogenesis to meet the demand for increased protein synthesis. Ribosome biogenesis, one of the most energy demanding processes, takes place in nucleoli. Notably, changes in number, size and morphology of nucleoli have been long recognized as a reliable feature of cancer cells. Interestingly, the nucleolus is well known to be a hub in the stress response. Approximately 70% of the ~4500 nucleolus-associated proteins are involved in functions other than ribosome biogenesis, including cell cycle control, apoptosis or DNA repair. This places the nucleolus as an important integrator between ribosome synthesis, cell cycle progression and stress signaling. A large variety of cellular insults trigger alterations in the dynamics of nucleolar proteins and/or ribosomal RNA. Stabilization of the tumor suppressor p53 has been observed as a result of the relocalization of certain ribosomal proteins in the nucleoplasm, notably L5 and L11, where they can bind and inhibit MDM2.

The aim of this work is to identify new potential anti-tumoral drugs by means of disrupting the nucleolar integrity. By using a high content cell-based screening in which nucleolar morphology is monitored by GFP-L37, we have identified several compounds that cause nucleolar disruption thereby leading cancer cells to proliferative arrest or apoptosis. Interestingly, one of our best hits is compound 2,4,7,9-tetramethylbenzo[b][1,8]naphthyridin-5-amine (TMBNA). This compound has been previously reported by other investigators to intercalate DNA and to activate p53, although the underlying molecular pathway is unknown. Our data indicates that TMBNA activates p53 through nucleolar disruption and binding of the ribosomal protein L11 to MDM2, ultimately resulting in cell cycle arrest and apoptosis. These results highlight the relevance of the nucleolus as a main target of cancer drugs.

Citation Format: Lucia Morgado-Palacin, Susana Llanos, Carmen Blanco-Aparicio, Diego Megias, Joaquin Pastor, Manuel Serrano. A cell-based screening to identify nucleolar disruptors in cancer cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B45.
Abstract B54: Squamous cell carcinoma antigen 1 (SCCA1) promotes mammary tumorigenesis through the induction of misfolded protein stress
Authors: Sheshadri Namratha , Ullman Erica , Catanzaro Joseph , Pan Ji-An , Chen Emily , Zong Wei-Xing
Published: August 2014

SCCA1, a member of the serine/cysteine protease inhibitor family, is upregulated in several cancers such as the cervix, lung, head and neck, liver, skin, and breast, and has been used as a diagnostic/prognostic marker. However, the biological function of SCCA1 in tumorigenesis remains largely unclear. Here we show that ectopic expression of SCCA1 in the non-tumorigenic mammary epithelial cell line MCF10A leads to spindle shaped cell morphology, increased cell migration, resistance to anoikis, foci formation in soft agar, as well as orthotopic tumor formation in immune-compromised mice. Gene expression analysis revealed that SCCA1 induces changes that are associated with epithelial-mesenchymal transition (EMT). Mechanistically, SCCA1 interferes with cellular protein homeostasis leading to a heightened unfolded protein response including the nuclear translocation of the transcriptionally active form of ATF6α. This increased ATF6α signaling results in NF-κB activation and increased pro-inflammatory response including an IL-6 autocrine signaling loop that drives EMT and transformation. These findings indicate a previously unknown role of SCCA1 in promoting tumorigenesis by inducing misfolded protein stress, NF-κB activation, and pro-tumorigenic cytokine signaling.

Citation Format: Namratha Sheshadri, Erica Ullman, Joseph Catanzaro, Ji-An Pan, Emily Chen, Wei-Xing Zong. Squamous cell carcinoma antigen 1 (SCCA1) promotes mammary tumorigenesis through the induction of misfolded protein stress. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B54.
Abstract B60: Interleukin-1 expression and inflammasome activation in human malignant glioma
Authors: Tarassishin Leonid , Lim Jihyeon , Lee Sunhee C.
Published: August 2014

Introduction: Interleukin 1 (IL-1) is a major proinflammatory cytokine produced by myeloid cells and is implicated in neurodegeneration. In human brain, IL-1 is expressed by microglia and contributes to neuroinflammation and neuronal toxicity. IL-1 is also the major activator of human astrocyte and glioma cells. In this study, we investigated the mechanism of IL-1 α and IL-1β expression and activation in human glioma cells.

Methods: Human glioblastoma cell lines (U87, U251, and SN19) as well as patient-derived glioma cell lines were stimulated with cytokines (IL-1/IFNγ) or TLR ligands (LPS or poly IC) and the expression of IL-1α and IL-1β was studied by real-time PCR, western blot analysis, and ELISA. The glioma secretome was investigated using quantitative mass spectrometry.

Results: We found that while primary human astrocytes show IL-1 translational block (no protein made despite high level of mRNA), but GBM cells responded strongly to IL-1 itself to produce large amounts of IL-1α and IL-1β mRNA and proteins. Furthermore, GBM cells processed pro-IL-1β (31kDa) to mature IL-1β (17kDa) by activating the NALP3 inflammasome. Both ATP and nigericin induced IL-1β processing by upregulating NALP3. Proteomics analysis of IL-1 stimulated GBM cell secretome revealed several highly upregulated proteins (IL-8, MCP-1, tenascin-C, pentraxin 3, galectin 1, and MMP2), all of which are implicated in glioma progression. Furthermore, many extracellular matrix proteins (ECM) and ECM-related proteins were downregulated by IL-1 in the glioma secretome.

Discussion: We propose that the ability of malignant glioma cells to produce IL-1 protein can set off chronic smoldering inflammation and induce epithelial-mesenchymal transition (EMT) resulting in increased migratory capacity, a unique gene signature, and increased immune signaling (NF-κB, pStat3). There is potential for IL-1 to be a biomarker/surrogate marker for a subset of GBM and that IL-1 blockade could benefit GBM patients.

Citation Format: Leonid Tarassishin, Jihyeon Lim, Sunhee C. Lee. Interleukin-1 expression and inflammasome activation in human malignant glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B60.
Abstract B67: Elucidating the mechanism of targeting WNT ligands to exosomes
Authors: Zhang Ying Yi , Underhill Ainsley , Zhang Liang , Wrana Jeffrey
Published: August 2014

It has been demonstrated that secreted vesicles called exosomes derived from stromal fibroblasts pick up Wnt ligands from breast cancer cells and facilitate cell migration and metastasis in vivo. However, how Wnt ligands are loaded onto exosomes remains unclear. Interestingly, Wnt ligands are posttranslationally modified by acylation and thus tether tightly to the cell membrane. I will present my work on defining the molecular mechanisms through which Wnt is targeted to exosomes. In particular, I am studying the molecular determinants in Wnt ligands, including the palmitoylation and glycosylation sites that regulate Wnt sorting to exosome membranes. Elucidation of Wnt mobilization in the extracellular space has significant implications in the fields of Wnt signaling in both development and cancer.

Citation Format: Ying Yi Zhang, Ainsley Underhill, Liang Zhang, Jeffrey Wrana. Elucidating the mechanism of targeting WNT ligands to exosomes. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B67.
Abstract C06: Translation of isoform-level gene signature into robust diagnostic assay for glioblastoma subtyping
Authors: Bi Yingtao , Pal Sharmistha , Macyszyn Luke , O'Rourke Donald , Davuluri Ramana
Published: August 2014

Abstract: Translating the molecular signatures from classification strategies into robust diagnostic assays is essential for cancer therapy guidance and prognosis determination. To develop a clinically applicable molecular stratification assay for Glioblastoma multiforme (GBM) with significant prognostic value, we designed a novel classification system based on isoform-level gene expression profiles; an avenue unexplored for diagnostic and prognostic use. Using isoform-level expression clustering of The Cancer Genome Atlas (TCGA) samples, we identified four GBM subgroups with significant (p=0.0103) survival differences. A four-class classifier, built with 121 transcript-variants, assigns GBM patients' molecular subtype with 92% accuracy. The classifier was translated to a high-throughput RT-qPCR assay and validated on an independent cohort of 206 glioblastoma samples. We found that proneural patients have the worst prognosis except for younger patients (<40 years), while a better prognosis is observed for neural group among older patients (≥40 years). The isoform-level classifier, transformed from a high-dimensional platform to a low-dimensional RT-qPCR platform, provides a quantitative and reproducible stratification of GBM patients with prognostic significance, a requirement towards personalized medicine. The resulting diagnostic assay from the classifier has immediate clinical implications towards prioritizing patients for standard care versus aggressive therapy regimen.

Citation Format: Yingtao Bi, Sharmistha Pal, Luke Macyszyn, Donald O'Rourke, Ramana Davuluri. Translation of isoform-level gene signature into robust diagnostic assay for glioblastoma subtyping. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C06.
Abstract C09: Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to cisplatin and poly (ADP-ribose) polymerase inhibition
Authors: Cavallo Francesca , Graziani Grazia , Antinozzi Cristina , Feldman Darren R. , Houldsworth Jane , Bosl George J. , Chaganti Raju S. K. , Moynahan Mary Ellen , Jasin Maria , Barchi Marco
Published: August 2014

Testicular Germ Cell Tumors (TGCT) and patient-derived cell lines are extremely sensitive to cisplatin and other interstrand cross-link (ICL) inducing agents. Nevertheless, a subset of TGCTs are either innately resistant or acquire resistance to cisplatin during treatment. Understanding the mechanisms underlying TGCT sensitivity/resistance to cisplatin as well as the identification of novel strategies to target cisplatin-resistant TGCTs have major clinical implications. Herein, we have examined the proficiency of five embryonal carcinoma (EC) cell lines to repair cisplatin-induced ICLs. Using γH2AX staining as a marker of double strand break formation, we found that EC cell lines were either incapable of or had a reduced ability to repair ICL-induced damage. The defect correlated with reduced Homologous Recombination (HR) repair, as demonstrated by the reduction of RAD51 foci formation and by direct evaluation of HR efficiency using a GFP-reporter substrate. HR-defective tumors cells are known to be sensitive to the treatment with poly(ADP-ribose) polymerase (PARP) inhibitor. In line with this observation, we found that EC cell lines were also sensitive to PARP inhibitor monotherapy. The magnitude of sensitivity correlated with HR-repair reduced proficiency and with the expression levels and activity of PARP1 protein. In addition, we found that PARP inhibition strongly enhanced the response of the most resistant EC cells to cisplatin, by reducing their ability to overcome the damage. These results point to a reduced proficiency of HR repair as a source of sensitivity of ECs to ICL-inducing agents and PARP inhibitor monotherapy, and suggest that pharmacological inhibition of PARP can be exploited to target the stem cell component of the TGCTs (namely ECs) and to enhance the sensitivity of cisplatin-resistant TGCTs to standard treatments.

Citation Format: Francesca Cavallo, Grazia Graziani, Cristina Antinozzi, Darren R. Feldman, Jane Houldsworth, George J. Bosl, Raju S. K. Chaganti, Mary Ellen Moynahan, Maria Jasin and Marco Barchi. Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to cisplatin and poly (ADP-ribose) polymerase inhibition. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C09.
Abstract C11: Molecular mechanism driving BMI1-induced melanoma metastasis
Authors: Ferretti Roberta , Lees Jacqueline A.
Published: August 2014

Malignant melanoma is one of the most aggressive human cancers, with a high potential for lethal metastasis and therapeutic resistance. The epigenetic chromatin regulator Bmi1 acts as a key component of the Polycomb Repressor Complex 1 (PRC1) and is a known oncogene. Various studies shown that BMI1 is highly expressed in many human hematopoietic malignancies and in different solid tumor types including prostate, breast, ovarian, colon, brain and lung cancer. Importantly, high Bmi1 expression is an excellent predictor of both metastatic progression and poor therapeutic response. Bmi1's role in tumor onset has been assessed using germline mutant mice showing that Bmi1 ablation efficiently suppress tumor development. This impaired tumorigenicity correlates with an impairment of the self-renewal and proliferative properties of tumor initiating cells, mainly due to Bmi1's repressive activity on the Ink4a/Arf tumor suppressor locus. Nevertheless, this function does not explain why Bmi1 is frequently upregulated in tumors nor Bmi1's role in tumor progression.

To evaluate Bmi1's contribution to melanoma progression, we upregulated Bmi1 in two different melanoma cell lines, a murine metastatic cell line and a poorly metastatic human cell line. We found that Bmi1 overexpression activates or increases invasive behavior of both cell lines, without enhancing their proliferative capacity. This finding allowed us to dissect Bmi1's oncogenic activity at later stages of tumorigenesis, in the absence of Bmi1's deregulation of cell proliferation. Our data show that Bmi1 significantly enhances in vitro cell movement and modulates adhesion. Bmi1 also protects cells from apoptosis induced by different stimuli and increases lung metastasis formation in vivo.

To characterize Bmi1-induced changes in gene expression, we have performed comparative deep sequencing of total RNA libraries generated from Bmi1 or GFP overexpressing cells. Gene ontology analysis revealed enrichment in cancer-relevant pathways that seem to be controlled by Bmi1, including cell movement, cell death and survival, tissue development and cell morphology. In particular, Bmi1 expression modulates the gene regulatory programs associated with EMT, TGFβ and the non-canonical Wnt pathways. We have identified a new Bmi1 target gene, poorly characterized, that seems to be responsible for Bmi1-induced melanoma progression. These data determine a causal role for Bmi1 in melanoma cell migration and establishment of distant melanoma metastasis.

Citation Format: Roberta Ferretti, Jacqueline A. Lees. Molecular mechanism driving BMI1-induced melanoma metastasis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C11.
Abstract C15: Therapeutic benefit of targeting ERK in mutant KRAS in pancreatic ductal adenocarcinoma (PDAC)
Authors: Hayes Tikvah K. , Neel Nicole F. , Chenard Melissa , Yeh Jen Jen , Samatar Ahmed , Der Channing J.
Published: August 2014

The limited success of anti-MEK and anti-RAF therapies with KRAS mutant cancers has been attributed, in part, to de novo or acquired resistance mechanisms that arise after prolonged treatment. A majority of resistance mechanisms involve kinome reprograming to reactivate ERK downstream of inhibitor activity. Therefore, we hypothesized that directly targeting ERK would be an effective method for overcoming these resistance mechanisms. To address this possibility, we evaluated the sensitivity of a panel of mutant KRAS pancreatic ductal adenocarcinoma (PDAC) cell lines to the highly selective SCH772984 ERK1/2 protein kinase inhibitor. First, we found that the anchorage-dependent growth of 6 of 12 cell lines were sensitive to SCH772984 treatment (IC50<2 μM). Some sensitive PDAC lines were resistant to the AZD6244 MEK1/2-selective inhibitor, indicating different mechanisms of action of these two inhibitors. Second, we identified phospho-AKT as a potential biomarker for PDAC SCH772984 sensitivity. Third, we assessed the molecular basis for anchorage-dependent growth inhibition. After 72 h treatment, flow cytometry analyses indicated impaired cell cycle progression through G1. Additionally, we found increased apoptosis as measured by formation of cleaved caspase-3. Together these observations suggest that sensitivity is a result of both impaired cell cycle progression and induction of apoptosis. Surprisingly prolonged SCH772984 treatment (2 weeks) resulted in senescence, as sensitive lines stained positive for senescence-associated beta-galactocidase. Furthermore, we found that senescent cells induced p16 and lost phospho-RB two common markers associated with induction of senescence. Onset of senescence was dependent on the loss of total MYC protein expression. Finally, we found that treatment with SCH772984 reduced tumorigenic growth in an in vivo xenograft mouse model. Our ongoing studies include determining the mechanisms of inhibitor resistance. In summary, our findings suggest that targeting ERK will be therapeutically beneficial for the treatment of KRAS mutant PDAC.

Citation Format: Tikvah K. Hayes, Nicole F. Neel, Melissa Chenard, Jen Jen Yeh, Ahmed Samatar, Channing J. Der. Therapeutic benefit of targeting ERK in mutant KRAS in pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C15.
Abstract C16: Mapping synthetic vulnerabilities in non-small cell lung cancer
Authors: Kim Hyun Seok , Mendiratta Saurabh , Kim Jiyeon , Pecot Chad Victor , Larsen Jill E. , Zubovych Iryna , Seo Bo Yeun , Kim Jimi , Eskiocak Banu , Chung Hannah , McMillan Elizabeth , Wu Sherry , Brabander Jef De , Komurov Kakajan , Posner Bruce A. , Brekken Rolf , Sood Anil K. , Deberardinis Ralph J. , Roth Michael G. , Minna John D. , White Michael A.
Published: August 2014

Diversity in the genetic lesions that drive cancer initiation and progression is extreme. This diversity exists not only among tumors from different patients, but also among cancer cells within the same patient. This nefarious complexity is, in large measure, responsible for the capacity of this disease to evade current best efforts for effective therapy. “Personalized medicine” has been proposed in response to this conundrum as a mechanism to tailor cancer treatment to a specific tumor's genetic and epigenetic characteristics. However, selection of appropriate treatment is dramatically limited by the paucity of appropriate drugs and by the difficulty of linking treatment options to the appropriate patients. The challenge is to identify authentic intervention targets for development of an appropriately diverse cohort of therapies to contend with disease heterogeneity. We are addressing this challenge by a focused investigation of common vulnerabilities that arise as a consequence of oncogene expression and tumor evolution. Here we will describe a cancer intervention discovery pipeline using parallel genetic and chemical perturbations within an extensive panel of cell lines representative of the molecular lesions detected in lung cancer by national and international cancer genome sequencing efforts. We have found that current first line targeted therapies are discoverable within this panel together with the enrollment biomarkers required to stratify patient treatment regimens. Further, we have found that new genetic and chemical vulnerabilities can be revealed that are linked to recurrent mutations in lung cancer patients that are not currently “actionable”. We are leveraging this approach to stratify lung cancer subtypes and elaborate intervention targets that are linked to those subtypes by robust molecular discriminators.

Note: This abstract was not presented at the conference.

Citation Format: Hyun Seok Kim, Saurabh Mendiratta, Jiyeon Kim, Chad Victor Pecot, Jill E. Larsen, Iryna Zubovych, Bo Yeun Seo, Jimi Kim, Banu Eskiocak, Hannah Chung, Elizabeth McMillan, Sherry Wu, Jef De Brabander, Kakajan Komurov, Bruce A. Posner, Rolf Brekken, Anil K. Sood, Ralph J. Deberardinis, Michael G. Roth, John D. Minna, Michael A. White. Mapping synthetic vulnerabilities in non-small cell lung cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C16.
Abstract C18: Interpreting the cancer genome using the Mouse Tumor Biology (MTB) Database
Authors: Krupke Debra M. , Begley Dale A. , Neuhauser Steven B. , Richardson Joel E. , Sundberg John P. , Bult Carol J. , Eppig Janan T.
Published: August 2014

For over a century cancer biologists have utilized the laboratory mouse in the study of cancer biology. Many mouse models recapitulate the genetic abnormalities observed in human cancers. The Mouse Tumor Biology Database (MTB; http://tumor.informatics.jax.org) provides multiple tools to explore and interpret the cancer genome. These tools include the ability to search MTB using mouse or human gene symbols, a mouse cancer quantitative trait loci (QTL) viewer, links to gene expression data for a variety of mouse models of human cancer, and access to public genomic data from the emerging Jackson Laboratory (JAX; http://www.jax.org) Patient-Derived Xenograft (PDX) program.

MTB was established in 1997 and provides freely available access to data on spontaneous and induced tumors from genetically defined mice (inbred, hybrid, mutant, and genetically engineered strains). These data include standardized tumor names and classifications, pathology reports and images, mouse genes and QTL associated with cancers, genomic and cytogenetic changes occurring in the tumor, strain names, tumor frequency and latency, and literature citations. Although the primary source for data represented in MTB is peer-reviewed scientific literature an increasing amount of data are derived from large systematic programs (e.g. JAX Aging Resource, PDX Program) and laboratory or consortia contributions. MTB includes annotated histopathology images and cytogenetic assay images for mouse tumors where these data are available. MTB encourages direct submission of mouse tumor data and images from the cancer research community and provides investigators with a web-accessible tool for image submission and annotation.

MTB is supported by NCI grant CA089713.

Citation Format: Debra M. Krupke, Dale A. Begley, Steven B. Neuhauser, Joel E. Richardson, John P. Sundberg, Carol J. Bult, Janan T. Eppig. Interpreting the cancer genome using the Mouse Tumor Biology (MTB) Database. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C18.
Abstract C31: Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models
Authors: Nygaard Vigdis , Prasmickaite Lina , Vasiliauskaite Kotryna , Clancy Trevor , Hovig Eivind
Published: August 2014

Melanomas are particularly renowned for their plasticity in response to microenvironmental signals. The brain offers a unique microenvironment to which malignant melanoma frequently spreads leading to dismal prognosis for the patients. The aim of our study was to reveal characteristics of metastatic growth of melanoma in the brain by in vivo imaging, cellular characterization and molecular approaches. The study was based on experimental metastasis models in vivo established from the melanoma cell lines, Melmet 1 and Melmet 5 with opposing phenotypes (invasive/proliferative, respectively) in the in vitro setting and distinct metastatic patterns in vivo. Gene expression profiling of brain metastatic cells isolated from different growth phases revealed the dynamic and opportunistic nature of melanoma in this context. We detected transitioning from the invasive phenotype to the proliferative phenotype, as the brain metastases passed from early to late growth phase. We also identified the existence of a neuron-like phenotype which included synaptic characteristics and a neurotransmission-like circuit involving glutamate receptors. This brain-adaptive phenotype was more prominent in the earlier metastatic growth phases compared to a later phase. To uncover microenvironmental factors that could play a role in melanoma cell colonization of the brain, we analyzed gene expression in host stroma. Genes implicated in immune-/inflammatory-responses were up-regulated and cellular characterization of metastatic brain tissue revealed the emergence and accumulation of cells of bone-marrow origin like microglia (F4/80+, CD11b+) that correlated with “active growth” phase initiation. Altogether, this suggests that acquisition of the brain-adaptive phenotype in a cooperative inflammatory environment might be critical for the establishment of metastatic growth in the brain microenvironment. Global network modeling and functional annotation analysis revealed that several of the central molecular factors and pathways mediating melanoma brain metastasis in this study, are also central to other pathological diseases, in particular neurodegenerative diseases. The identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets.

Citation Format: Vigdis Nygaard, Lina Prasmickaite, Kotryna Vasiliauskaite, Trevor Clancy, Eivind Hovig. Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C31.
Abstract C40: Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells
Authors: Sobol Anna , Galluzzo Paola , Liang Shuang , Rambo Brittany , Skucha Sylvia , Weber Megan , Bocchetta Maurizio
Published: August 2014

In previous studies we targeted hypoxic Non-Small Cell Lung Cancer (NSCLC) tumor tissue using gamma-secretase inhibitors (GSI) in a preclinical model of advanced NSCLC. GSI treatment of mice suggested reactivation of mTORC-1 in otherwise quiescent hypoxic NSCLC tissue.

Methods included immunohistochemistry and cytochemistry, immunoblotting, immunoprecipitation, siRNA-mediated gene knockdown, nucleotide and amino acid analogs incorporation, FACS analysis, various imaging techniques, Q-PCR, gene transfection, cap binding assays, RNA and antibodies arrays.

GSI treatment of mice showed a re-induced phosphorylation of 4E-BP1 at T37/46 in hypoxic tumor masses. In vitro studies indicated a role for APP in this phenomenon. APP downregulation caused a rearrangement in 4E-BP1's phosphorylation pattern at residues T37/46, S65 and T70, reversed by overexpression of the APP C-terminal domain. Increased recruitment of eIF4A on the mRNA cap was observed. These events coincided with a drastic increase in the rate of global protein synthesis, an event that appeared to be mTOR-independent.

Meanwhile, NSCLC cells with depleted APP were arrested in G0/G1 through a cyclin C-mediated mechanism. Cells arrested in G0/G1 with augmented rate of global protein synthesis (both cap- and IRES-dependent) increased their size and underwent a substantial necrotic cell death due to cell membrane permeabilization.

These results indicate a novel role for APP in synchronizing the rate of protein synthesis and cell cycle progression, raising questions on how these processes interact in malignant transformation.

The data presented here suggest that cells of different origin, including NSCLC cells, have developed some mechanisms to couple cell growth with cell cycle progression independent of overlapping signals which derive, among others, from the PI3-K pathway. APP seems to play a central role in this synchronization, and its loss of function causes cell size abnormalities and death.

Citation Format: Anna Sobol, Paola Galluzzo, Shuang Liang, Brittany Rambo, Sylvia Skucha, Megan Weber, Maurizio Bocchetta. Amyloid precursor protein (APP) synchronizes cell cycle progression and the rate of global protein synthesis in dividing cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C40.
Abstract C46: The pharmacokinetics and pharmacodynamics study of honokiol in Sprague-Dawley rats and colorectal tumor-bearing mice
Authors: Tsai Yung-Jen , Tsai Pei-Yi , Hung Le-Mei , Su Mei-Yin , Lin Min-Hui , Kuan Lung-Yu , Tsai Yih-Chiao , Chen Hui-Ling , Chang Jia-Ming
Published: August 2014

Honokiol, a small-molecule pharmacological bioactive constituent of the bark of Magnolia plants, has shown anti-tumor effect in a variety of human cancer xenograft models through the induction of apoptosis. Additionally, honokiol could enhance the cytotoxicity in combination of oxaliplatin in colon cancer cells.

To investigate the anti-tumor effect and the pharmacokinetics (PK) of honokiol, honokiol was tested for anti-tumor activity in a colorectal cancer xenograft mice model and oral-bioavailability of honokiol was assayed in Sprague-Dawley rats.

The pharmacokinetic results of 5.0 mg/kg honokiol after intravenous injection showed that the elimination half time was about 0.79 ± 0.03 hours, the Cmax was about 752.67 ± 58.36 ng/mL, and the mean residence time (MRT) was about 0.85 ± 0.05 hours.

In vivo study for testing anti-cancer activity of honokiol is underwent and expected that honokiol would inhibit the tumor growth in colorectal tumor-bearing mice. In conclusion, honokiol may be developed be as therapeutic agent for cancer treatment in combination with PK and pharmacodynamics (PD) data.

Citation Format: Yung-Jen Tsai, Pei-Yi Tsai, Le-Mei Hung, Mei-Yin Su, Min-Hui Lin, Lung-Yu Kuan, Yih-Chiao Tsai, Hui-Ling Chen, Jia-Ming Chang. The pharmacokinetics and pharmacodynamics study of honokiol in Sprague-Dawley rats and colorectal tumor-bearing mice. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C46.