bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1100: Macrophage infiltration is associated with release from dormancy in a novel model of dormant prostate cancer
Authors: Long Thomas , Sprenger Cynthia C. , Coleman Ilsa , Nelson Peter , Ratner Buddy , Plymate Stephen
Published: May 2015

Background: Circulating tumor cells are found in approximately 70% of men with prostate cancer at the time of primary treatment. Less than a third of these men will develop recurrent cancer within 20 years of primary treatment, suggesting that only a subset of dormant tumor cells are able to escape dormancy and develop into clinical metastases. Escape from dormancy, though, is clinically significant since these men are at greatest risk of dying from prostate cancer. Mechanisms by which cancer cells escape dormancy are poorly understood and very few models exist to study dormancy, especially in prostate cancer. We previously found that when a dormant prostate cancer cell line was seeded into a bioscaffold and placed in vivo, tumor formation was detected after 6-8 weeks, whereas no growth was seen up to six months after the dormant cells were injected s.c. with Matrigel. Analysis of the tumors revealed significantly greater macrophage infiltration into the scaffold tumors. Gene expression and cytokine antibody arrays demonstrated increased expression of macrophage associated cytokines in scaffold tumors. While macrophages have been implicated in metastatic spread of various cancers, their role in escape from dormancy has not been elucidated.

Objective: To determine if macrophages play a role in escape from tumor dormancy in prostate cancer.

Methods: In vitro we cultured a dormant prostate cancer cell line, M12mac25, with macrophage conditioned media and then examined proliferation and gene expression. In vivo, mice were implanted with sphere-templated polyHEMA bioscaffolds pre-seeded with M12mac25 cells. To inhibit macrophage activity, mice received s.c. injections of clondronate liposomes or PBS liposomes as a control for 6 weeks.

Results: In vitro culture of M12mac25 cells with macrophage-derived conditioned medium led to increased proliferation of the M12mac25 cells and similarly upregulated genes as those seen the in vivo bioscaffold-derived tumors. In vitro treatment of primary bone-marrow derived macrophages with clondronate liposomes resulted in apoptosis while exposure to PBS liposomes did not affect the macrophages. In contrast, M12mac25 cells were not affected by either PBS or clondronate liposomes. In vivo, clondronate liposomes effectively eliminated macrophages from the M12mac25 seeded scaffolds and prevented tumor growth in these scaffolds.

Interpretation: Macrophages contribute to M12mac25 escape from dormancy.

Citation Format: Thomas Long, Cynthia C. Sprenger, Ilsa Coleman, Peter Nelson, Buddy Ratner, Stephen Plymate. Macrophage infiltration is associated with release from dormancy in a novel model of dormant prostate 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 1100. doi:10.1158/1538-7445.AM2014-1100
Abstract 1104: The role of DLL4 in cancer immune-surveillance and its pathogenicity in p53-dysregulated environment
Authors: Yao Zhixing , Sherif Zaki A.
Published: May 2015

Recent advances in cancer studies reveal that tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome characterized by mutation of the TP53 tumor suppressor gene (locus 17p13.1). The p53 transcription factor responds to diverse cellular stresses by regulating target genes that induce apoptosis, cell cycle arrest, DNA repair, and senescence. LFS can provide powerful insights into our understanding of somatic mutations present in sporadic cancers as well as the de-regulation of cell signaling pathways. We will be using LFS cells and mice as models to build on our previous discovery that showed a balanced reciprocal translocation between chromosomes 11q23 and 15q15 in LFS patients' skin fibroblast cells. In our present study, analysis of the breakpoint region reveals that in LFS, breast cancer (MCF7) and neuroblastoma (IMR32) cell lines, there is a dramatically increased activation in the expression of cancer susceptibility candidate 5 gene (CASC5), a part of kinetochore-microtubule attachments associated with cell proliferation. Furthermore, there was an abrogation in the expression of a critical protein, Delta-like ligand-4 (DLL4), in LFS normal skin fibroblasts of TP53 mutation carriers and non-carriers. There was also a down-regulation of DLL4 in unrelated breast cancer and neuroblastoma cell lines by DNA hypermethylation. DLL4 is a ligand for Notch family of receptors with crucial functions in normal development, immune surveillance and tissue homeostasis. TP53 and CTCF (a transcription factor involved in chromatin architecture) have a direct activator effect at the DLL4 promoter. Notch/DLL4 plays a key role in the generation of hematopoietic stem cells during embryonic development and regulates the T cell lineage and subsequent stages of thymopoiesis. WNT2 is also down-regulated in LFS cell lines and abolished in MCF7. Whereas Notch signaling controls cell fate specification in a variety of cell contexts during embryonic and postnatal development, Wnt signaling plays a critical role in cell differentiation and proliferation. Both Notch and Wnt pathways are activated by oncogenic mutations and dysregulated in cancer. The long-term objective of this research is to develop a critical understanding of the mechanisms by which p53 regulates cancer immune-surveillance through cross-talk with the tumor microenvironment so that therapeutic agents can be developed to curtail cancer progression.

Citation Format: Zhixing Yao, Zaki A. Sherif. The role of DLL4 in cancer immune-surveillance and its pathogenicity in p53-dysregulated environment. [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 1104. doi:10.1158/1538-7445.AM2014-1104
Abstract 1106: The tumor immune microenvironment modulates response to chemotherapy in high-grade serous epithelial ovarian cancer
Authors: Koti Madhuri , Edwards Andrew , Squire Jeremy A.
Published: May 2015

Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. Recent studies have drawn attention to the role of the tumor-microenvironment in mediating chemotherapy resistance, in addition to established mechanisms of tumor progression. This project is investigating the role of the tumor inflammatory microenvironment in facilitating intrinsic chemotherapy resistance using a cohort of 36 high-grade serous epithelial ovarian cancer patient tumor samples. Total RNA from forty-eight, HGSEOC tumor tissue samples was subjected to gene expression profiling using NanoString technology. This type of bar-coded expression profiling has proven to be equally efficient to study gene expression as quantitative real time PCR technique. The gene panel consisted of 184 human genes that are differentially expressed in human inflammation and 6 internal reference genes. The cohort comprised 18 sensitive (PFS >18months) and 18 resistant (PFS<6 months) samples. A multiplex cytokine assay using the bioplex system was also performed on serum samples from a subset of this cohort. The results showed significant differences in expression of some inflammatory cytokines in the serum that were concurrent with the observed gene expression differences in the tumor samples. The results are indicative of a cooperative role of a specific inflammatory gene signature within the tumor microenvironment that may augment pathways leading to differential drug response. These studies need further independent validations to derive non-invasive blood-based biomarkers for use in predicting chemotherapy resistance in serous epithelial ovarian cancer.

Citation Format: Madhuri Koti, Andrew Edwards, Jeremy A. Squire. The tumor immune microenvironment modulates response to chemotherapy in high-grade serous epithelial 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 1106. doi:10.1158/1538-7445.AM2014-1106
Abstract 1129: Adenylate kinase 4 promotes metabolic reprogramming and induces EMT in lung cancer through ROS-dependent stabilization of HIF1-α
Authors: Jan Yi-Hua , Hsiao Michael
Published: May 2015

The metabolic alterations that are essential for cancer progression remain largely unknown. Previously, we have identified adenylate kinase 4 (AK4) as a marker for poor clinical outcome that promotes metastasis of lung cancer. However, the molecular mechanism of AK4-induced phenotype remains elusive. By microarray analysis, we found downstream targets of HIF1-α were differentially regulated upon AK4 overexpression in CL1-0 lung cancer cells. Furthermore, AK4 overexpression induces glycolysis and results in accelerated glucose consumption, ATP utilization and lactate production. Overexpression of AK4 also increases the levels of reactive oxygen species (ROS), which exaggerates HIF1-α protein expression under hypoxia and concurrently induces epithelial-to-mesenchymal transition (EMT) in a HIF1-α-dependent manner. IHC analysis showed an inverse correlation between AK4 and E-cadherin and a positive correlation between AK4 and nuclear HIF1-α in lung cancer patients. Moreover, patients with AK4 high/E-Cadherin low showed worse outcome compared to patients with AK4 low/E-cadherin high. Our study indicates that enhanced expression of AK4 is a critical factor to trigger aerobic glycolysis and induced HIF1-α-mediated EMT in lung cancer.

Citation Format: Yi-Hua Jan, Michael Hsiao. Adenylate kinase 4 promotes metabolic reprogramming and induces EMT in lung cancer through ROS-dependent stabilization of HIF1-α. [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 1129. doi:10.1158/1538-7445.AM2014-1129
Abstract 1130: AKT-mediated phosphorylation is responsible for TWIST1-mediated tumor growth and metastasis
Authors: Ozes Osman N. , Pehivanoglu Suray , Ertosun Gokhan , Gorgisen Gokhan , Erin Nuray , Unal Duygu , Tanriover Gamze
Published: May 2015

Cancer cells show epithelial-mesenchymal transition (EMT) during cell migration, invasion and dissemination. Although it has been shown by numerous publications that the evolutionary conserved basic helix-loop-helix (b-HLH) transcription factor TWIST1 plays pivotal role during EMT the molecular mechanism responsible for TWIST1 function is not fully understood. Here we show that Twist1 binds to and phosphorylated by AKT/protein kinase-B at S42, T121 and S123. While conversion of S42, T121 and S123 to phosphorylation-mimicking Aspartic or glutamic acids created active Twist1, Alanin mutants of the same sites diminished the DNA-binding and transctivating functions of Twist1. In line with this, Glutamic Acids mutants suppressed the expression of E-Cadherin, whose expression is negatively regulated by active TWIST1, Alanin mutants induced the expression of E-Cadherin. Similarly, we tested the impact of above mentioned mutants on cell migration and proliferation. Our results demonstrated that while Glutamic acid mutants accelerated, Alanin mutants suppressed the migration and proliferation of 293T cells. Our in vitro results prompted us to test our mutants under in vivo conditions using breast cancer as a model. We transfected non-metastatic mouse mammary tumor cell line, 67NR, with Glutamic acid mutants, and metastatic mouse mammary tumor cell line, 4T1, with Alanin mutants of mouse TWIST1 and selected the Neo-resistant populations. We injected 1 million 67NR cells over-expressing wild type or Glutamic acid mutants of Twist1, and 100.000 4T1 cells over-expressing wild type or Alanin mutants of Twist1 into the breast tissue of female inbred BalBC mice, and animals were monitored for metastasis. At the end of the 4th week, we sacrifised and examined the animals for tumor growth and metastasis. Although 67NR cells over-expressing wild type Twist1 did not show any metastasis, cells over-expressing 1E (S42E) and 2E (S42E, T121E) mutants of Twist1 showed 15-20 macroscopic metastasis to liver and Lungs. Paralel to this, 4T1 cells expressing 1A (S42A) and 2A (S42A, T121A) mutants of TWIST1 showed no macroscopic metastasis. Our results clearly indicate that phosphorylation of S42 and T121 by AKT is essential for TWIST1-mediated tumor growth and metastasis.

Citation Format: Osman N. Ozes, Suray Pehivanoglu, Gokhan Ertosun, Gokhan Gorgisen, Nuray Erin, Duygu Unal, Gamze Tanriover. AKT-mediated phosphorylation is responsible for TWIST1-mediated tumor growth and metastasis. [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 1130. doi:10.1158/1538-7445.AM2014-1130
Abstract 1146: Up-regulation of integrin β4 promotes epithelial-mesenchymal transition and is a novel prognostic marker in pancreatic cancer
Authors: Masugi Yohei , Yamazaki Ken , Emoto Katsura , Effendi Kathryn , Kitago Minoru , Itano Osamu , Kitagawa Yuko , Sakamoto Michiie
Published: May 2015

Pancreatic cancer is a highly aggressive and lethal malignant tumor. Recent reports have shown that the epithelial-mesenchymal transition (EMT) plays an essential role in cancer invasion and metastasis. We previously reported that EMT was frequently observed in pancreatic ductal adenocarcinoma (PDA) and was related to a high metastatic rate and a poor outcome in patients with PDA (Masugi et al. Hum Pathol. 2010). Further, using cDNA microarray analyses, we identified integrin β4 (ITGB4) as one of the EMT signature genes in 12 patient-derived PDA xenografts. The aim of the current study was to clarify the clinicopathological and functional significance of ITGB4 in PDA. ITGB4 up-regulation in xenografts with high EMT was confirmed by immunohistochemistry. Immunohistochemical analyses of 134 resected PDAs revealed intratumoral heterogeneity with respect to ITGB4 expression and showed that cancer cells undergoing EMT often display diffuse and intense expression of ITGB4. High levels of ITGB4 expression were significantly correlated with the histological hallmarks of EMT (solitary cell infiltration, reduced E-cadherin expression and increased vimentin expression), the grade, and the presence of lymph node metastasis, and showed an independent prognostic effect. Immunocytochemical analyses of PDA cell lines revealed that the distribution of ITGB4 changed from regions of cell-cell contact to a diffuse presence in cytoplasm with occasional localization in filopodia during EMT. Knockdown of ITGB4 by small interfering RNA reduced the migratory and invasive ability of PDA cells. Overexpression of ITGB4 promoted cell scattering and cell motility in combination with down-regulation of E-cadherin and up-regulation of vimentin expression. In conclusion, our study demonstrated the prognostic significance of ITGB4 overexpression in PDA. Moreover, we also identified a potential role for ITGB4 in the regulation of cancer invasion and EMT.

Citation Format: Yohei Masugi, Ken Yamazaki, Katsura Emoto, Kathryn Effendi, Minoru Kitago, Osamu Itano, Yuko Kitagawa, Michiie Sakamoto. Up-regulation of integrin β4 promotes epithelial-mesenchymal transition and is a novel prognostic marker in pancreatic 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 1146. doi:10.1158/1538-7445.AM2014-1146
Abstract 1147: CD44s is required for EMT and stemness properties via ZEB1 activation
Authors: Preca Bogdan-Tiberius , Bajdak Karolina , Pfannstiel Jessica , Brabletz Thomas
Published: May 2015

Invasion and metastasis of carcinomas are often promoted by the aberrant epithelial to mesenchymal transition (EMT) program at the invasive front of the tumor. The transcription factor ZEB1 is a crucial promotor of metastasis formation by inducing EMT and tumor initiating capacity. CD44, a cell surface protein, is used as marker to identify tumor initiating cells in many cancer types. It is involved in cell-cell interactions, cell adhesion and migration. Through alternative splicing, controlled by the epithelial splicing factor ESRP1, CD44 is differentially regulated during EMT, resulting in a switch from the variable exon-containing CD44v isoforms to the standard isoform CD44s, which is devoid of all CD44 variable exons.

We examine the reciprocal interaction of CD44s and ZEB1 in their function as tumor initiation markers. CD44s and ZEB1 are co-expressed in poorly differentiated human cancer cells.Transient down regulation of CD44s results in decrease of ZEB1, which in turn leads to reduced sphere formation ability. Likewise, inhibition of CD44s increases ESRP1. Knockdown of ESRP1 decreases expression of CD44v isoforms, increases CD44s and ZEB1 and the sphere forming capacity. Additional knockdown of CD44s reduces the sphere formation. Furthermore, ZEB1 inhibits ESRP1 expression, which leads to upregulation of CD44s.

In summary we identified a positive feedback loop maintaining stemness and mesenchymal features in cancer cells: CD44s activates expression of ZEB1, which suppresses transcription of the splicing factor ESRP1, resulting in further increase in CD44s expression.

Citation Format: Bogdan-Tiberius Preca, Karolina Bajdak, Jessica Pfannstiel, Thomas Brabletz. CD44s is required for EMT and stemness properties via ZEB1 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 1147. doi:10.1158/1538-7445.AM2014-1147
Abstract 1152: Gene profiling of multiple myeloma: MAPK pathway deregulation, which is regulated by PIM-1 and MOS, is associated with relapse within 6 months after autoSCT in MM patients
Authors: Jung Woo June , Ahn Kwang-Sung , Lee Chansu , Koh Youngil , Lee Hyun Jung , Kim Hyo Jung , Yoon Hwi-Joong , Yoon Sung-Soo
Published: May 2015

Substantial advances have been made in understanding the biology of multiple myeloma (MM) through the study of the bone marrow (BM) microenvironment. Dynamic interplay between bone marrow stromal cells (BMSCs) and MM cells regulated the relapsed MM after autologous bone marrow transplantation (ABMT). In this study, we performed gene expression profiling with microarray data to better dissect the molecular phenotypes and prognoses of relapsed multiple myeloma (MM). Using gene expression and clinical data, we applied gene expression signatures reflecting deregulation of oncogenic pathways to highlight molecular changes in bone marrow aspirate from 28 patients with relapsed MM. The patient subgroups were defined according to relapse-free interval, within 6 months versus more than 6 months. The microarray results revealed that dyregulation of MAPK pathway was associated with relapse within 6 months after ABMT. Among them, the expression of PIM-1 gene and MOS gene was higher in samples from patients with relapsed MM than MM cell lines (p=0.0037; p=0.0021). Also, IL-6, sIL-6R, and HGF expression in patients who relapsed within 6 months after ABMT was higher than the patients whose relapse-free interval were longer than 6 months. Treatment of shRNA PIM-1 gene and shRNA MOS gene in U266 and MOLP8 dramatically led to decreasing IL-6/sIL-6R mediated ERK phosphorylation and HGF-mediated MET phosphorylation. Similar results were noted for cluster genes for PIM-1/MOS. Especially, functional analysis of PIM-1 leads to inactivate the MAPK pathway through regulating p38 mediated signaling and Wnt/β-catenin pathway. Recent studies suggested that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Conclusionally, Our analysis suggested that MOS gene and PIM-1, which regulates MAPK pathway, is a noble prognostic marker for relapse of MM. The importance of the PIM-1-MOS-MAPK pathway as a prognostic marker in relapsed MM should be reassessed in the novel therapeutic agent era.

Citation Format: Woo June Jung, Kwang-Sung Ahn, Chansu Lee, Youngil Koh, Hyun Jung Lee, Hyo Jung Kim, Hwi-Joong Yoon, Sung-Soo Yoon. Gene profiling of multiple myeloma: MAPK pathway deregulation, which is regulated by PIM-1 and MOS, is associated with relapse within 6 months after autoSCT in MM patients. [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 1152. doi:10.1158/1538-7445.AM2014-1152
Abstract 1156: Shed syndecan-1 downregulates histone acetyltransferase activity and shuttles HGF to the nucleus of tumor and host cells
Authors: Stewart Mark , Ramani Vishnu , Sanderson Ralph
Published: May 2015

The cell surface heparan sulfate proteoglycan syndecan-1 (CD138) is expressed by myeloma tumor cells, is proteolytically shed from the cell surface and accumulates in the serum. High levels of serum syndecan-1 are an independent indicator of poor prognosis. Our lab has demonstrated that shed syndecan-1 drives myeloma tumor progression in vivo, but the mechanism(s) mediating this are not fully understood. The role of heparan sulfate proteoglycans on the cell surface and in the extracellular matrix has been extensively studied, but their presence and function within the nucleus is often overlooked. However, recent findings indicate that nuclear heparan sulfate has important regulatory functions. Based on preliminary findings, we hypothesized that shed syndecan-1 binds to the tumor cell surface and translocates to the nucleus where it regulates gene expression thereby promoting aggressive tumor behavior. We utilized murine myeloma cells which were grown in the presence of medium containing human shed syndecan-1. Strikingly, the shed syndecan-1 was rapidly taken up by these cells and translocated to the nucleus. Additionally, exogenous human shed syndecan-1 added to murine stromal cells also bound to the cell surface and translocated to the nucleus. This is the first demonstration that shed proteoglycans can translocate to the nucleus of cells. When stromal cells were grown in the presence of tumor-derived shed syndecan-1, acetylated histone H3 was decreased. Additionally, tumor cells engineered to express high levels of shed SDC1 have a 58% reduction in histone acetyltransferase activity. This may be due to syndecan-1 directly binding to p300/CBP-associated factor, a HAT enzyme. Moreover, HGF bound to shed syndecan-1 was shuttled to the nucleus of myeloma cells as a complex. Shed syndecan-1 and HGF translocation to the nucleus is dependent on intact heparan sulfate chains present on the syndecan-1 core protein. These data reveal a novel mechanism of tumor-host crosstalk whereby syndecan-1, shed by tumor cells, alters histone acetyltransferase activity and shuttles heparin-binding factors to the nucleus to reprogram cells and alter gene expression. Therapeutic regulation of syndecan-1 shedding, its binding to the cell surface or translocation to the nucleus represent novel strategies to control the progression of myeloma and other cancers.

Citation Format: Mark Stewart, Vishnu Ramani, Ralph Sanderson. Shed syndecan-1 downregulates histone acetyltransferase activity and shuttles HGF to the nucleus of tumor and host cells. [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 1156. doi:10.1158/1538-7445.AM2014-1156
Abstract 1164: sHER3 inhibits the proliferation and migration of melanoma-derived cells in a tenascin-dependent manner
Authors: Cai Chunlin , Maihle Nita J.
Published: May 2015

The expression of HER3 receptor tyrosine kinase, a member of the EGFR/HER family of cellular oncogenes, is common in both primary and metastatic melanoma, and high levels of HER3 expression are correlated with poor patient survival. However, no one has demonstrated the mechanistic basis for HER3 as a poor prognostic determinant in melanoma patients, even though these tumors poorly express other members of the EGFR/HER receptor family. In this study we present evidence demonstrating that 13 of 14 melanoma cell lines tested express HER3, supporting previous reports regarding the frequency of expression of this gene product in melanoma samples. We further report that a soluble isoform of HER3 (sHER3) that we previously have shown to be a potent inhibitor of neuregulin mediated, Akt-dependent breast cancer cell growth strongly inhibits the growth of HER3 expressing melanoma cells. Interestingly, however, the phosphorylation of Akt, a known mediator of ligand-dependent HER3 signaling, is not reduced in melanoma cells growth inhibited by sHER3. To explore alternative mechanisms of HER3 signaling in melanoma, we identified sHER3 interacting partners using a mass spectrometry proteomic approach. Only one prominent sHER3 binding partner was identified in melanoma cell conditioned media: tenascin. This was an unexpected finding since ligand-dependent HER3 signaling has not previously been linked to tenascin. Interestingly, tenascin expression has independently been identified as another negative prognostic indicator in melanoma. Our initial studies to test the functional relationship between HER3 and tenascin suggest that tenascin expression is correlated with both HER3 expression and migration in all of the melanoma lines tested. Moreover, sHER3 inhibition of melanoma migration was dependent on the level of tenascin present. Treatment of melanoma cells with purified sHER3 inhibits the migration only in the presence of high levels of tenascin. These results suggest the existence of an unanticipated tenascin- and HER3-dependent signaling pathway in melanoma cells, and suggest that this pathway is functionally correlated with melanoma cell migratory potential and poor patient survival. (This work was supported by the Harry J. Lloyd Charitable Trust Foundation.)

Citation Format: Chunlin Cai, Nita J. Maihle. sHER3 inhibits the proliferation and migration of melanoma-derived cells in a tenascin-dependent manner. [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 1164. doi:10.1158/1538-7445.AM2014-1164
Abstract 1181: The Apoptosis repressor with a CARD domain (ARC) is a direct HIF1 target gene and promotes survival and proliferation of VHL deficient renal cancer cells
Authors: Razorenova Olga V. , Castellini Laura , Colavitti Renata , Edgington Laura E. , Nicolau Monica , Huang Xin , Bedogni Barbara , Mills Edward M. , Bogyo Matthew , Giaccia Amato J.
Published: May 2015

The induction of hypoxia inducible factors (HIFs) is essential for the adaptation of tumor cells to a low oxygen environment. We found that the expression of the apoptosis inhibitor ARC was induced by hypoxia in a variety of cancer cell types and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at -190 bp upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel Lindau (VHL) gene. Accordingly, our results show that ARC is not expressed in normal renal tissue, but is highly expressed in 65% of RCC tumors, which also express high levels of Carbonic Anhydrase IX (CAIX), a HIF1-dependent gene. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

Citation Format: Olga V. Razorenova, Laura Castellini, Renata Colavitti, Laura E. Edgington, Monica Nicolau, Xin Huang, Barbara Bedogni, Edward M. Mills, Matthew Bogyo, Amato J. Giaccia. The Apoptosis repressor with a CARD domain (ARC) is a direct HIF1 target gene and promotes survival and proliferation of VHL deficient renal cancer cells. [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 1181. doi:10.1158/1538-7445.AM2014-1181
Abstract 1194: IDH1 mutant inhibitor induces cellular differentiation and offers a combination benefit with Ara-C in a primary human Idh1 mutant AML xenograft model
Authors: Ellwood-Yen Kate , Chen Yue , Wang Fang , Lemieux Rene , Popovici-Muller Janeta , Yang Hua , Straley Kimberly , Choe Sung , Dorsch Marion , Agresta Sam , Schenkein David , Biller Scott , Su Michael
Published: May 2015

Somatic point mutations in isocitrate dehydrogenase 1/2 have a gain-of-function neomorphic activity that converts alpha-ketoglutarate to the oncometabolite, R (-)-2-hydroxyglutarate (2HG). Prospective studies of AML patients carrying IDH mutations have shown that intracellular concentrations of 2HG can range from 3-10 mM. This abnormal level of 2HG results in dysregulation of alpha-ketoglutarate dependent enzymes leading to alterations in the epigenetic state of hematopoietic progenitor/stem cells and functionally blocks their ability to fully differentiate.

We have developed a potent and selective, orally available IDH1 mutant inhibitor AGI-14100, that is able to reduce intracellular 2HG concentrations to baseline levels found in wildtype cells.

We next treated a primary human IDH1 (R132H)/FLT3-ITD mutant xenograft model with AGI-14100 either alone or in combination with Ara-c. In these studies, AGI-14100 alone significantly decreased tumor burden in the peripheral blood after 1 month of continuous BID treatment. In combination with a short-term, low-dose course of Ara-C, we also observed a decrease in the bone marrow tumor burden that was better than either treatment alone. Furthermore, this response was sustainable for >3 weeks even after dosing of both drugs had been terminated. Taken together, these data suggest that inhibition of mutant IDH1with AGI-14100 and low dose Ara-c could provide a combination benefit for patients with AML.

Citation Format: Kate Ellwood-Yen, Yue Chen, Fang Wang, Rene Lemieux, Janeta Popovici-Muller, Hua Yang, Kimberly Straley, Sung Choe, Marion Dorsch, Sam Agresta, David Schenkein, Scott Biller, Michael Su. IDH1 mutant inhibitor induces cellular differentiation and offers a combination benefit with Ara-C in a primary human Idh1 mutant AML xenograft model. [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 1194. doi:10.1158/1538-7445.AM2014-1194
Abstract 1199: Isolation and characterization of human breast epithelial cells
Authors: Hoynowski Steven
Published: May 2015

Breast cancer is one of the most common cancers among women in the United States and it is the second leading cause of cancer deaths in this population. Roughly 232,000 women in the U.S. were diagnosed with malignant breast cancer in 2013 and approximately 40,000 of these patients will die. The majority of breast cancers originate in the lobular or ductal cells of the milk-producing glands. In these structures, there are two main cell types: the inner luminal cells, which are surrounded by basal myoepithelium. These cells are structurally distinct and are the precursors to variable forms of breast cancer. As such, it is important to study them not only separately but also together. Current methods for culturing human mammary epithelial cells select for those of a basal phenotype. Isolation of pure basal and luminal cell populations was accomplished by using antibody-linked magnetic beads to isolate essentially pure populations of both basal (CALLA/CD10) and luminal mammary (MUC-1/CD227) epithelial cells from organoid explant cultures. Organoids were isolated and plated on collagen I coated flasks in either DFCI (basal) or MBCM (luminal) media and cultured at 37°C in 5% CO2 to induce cell migration. The media was replenished every 2 - 3 days until the cells reached confluence. Cultured cells were harvested and immunopurified using a CALLA/CD10 or MUC-1/CD227 antibody and magnetic beads. Positively selected cells were seeded in tissue culture treated flasks and propagated with medium exchange every 2 - 3 days. Each cell population was analyzed using flow cytometry to determine their purity. The DFCI/CD10 cells were over 98% CD10 positive with very little MUC1 reactivity, suggesting that they are an essentially pure basal epithelial cell population. The cells isolated using MBCM/MUC1 appear to be a mixed population with 86% MUC1 positive luminal cells and the remainder being CD10 positive basal cells. A second round of immunopurification resulted in an isolated cell population that was greater than 95% MUC1 positive, suggesting a pure luminal epithelial cell population. This procedure has been used on several organoid preparations and has generated high cell yields in addition to high cell purity. These isolated cell types can now be tested individually for drug sensitivity.

Citation Format: Steven Hoynowski. Isolation and characterization of human breast epithelial cells. [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 1199. doi:10.1158/1538-7445.AM2014-1199
Abstract 1217: Development of a spontaneous in vivo cachexia model using the Champions TumorGraft™ platform
Authors: Anderson Nathan , Khor Tin , Feldhaus Andrew , Gatling Andreya , Olson Katie , Latham John , Sidransky David , Bruckheimer Elizabeth M.
Published: May 2015

Cachexia is present in the majority of patients with advanced cancer progression and is not reversible through nutritional supplementation, leading to loss of skeletal muscle and adipose tissue. There are a lack of quality pre-clinical models for cachexia research. Previously, Champions Oncology reported a mouse cachexia model derived from a primary human renal cell carcinoma tumor implanted in immunocompromised mice. This model, CTG-0804, was developed using the innovative Champions TumorGraft™ platform, which preserves the biological properties of the original human tumor. The Champions TumorGraft platform demonstrated dramatic weight loss in these mice as the tumor grew. Plasma from these mice also showed elevated levels of human IL-6. We have now further characterized this TumorGraft model by evaluating standard of care (SOC) agents for renal cell carcinoma and anti-IL-6 compounds to determine the mechanism of cachexia. The SOC evaluation revealed that sorafenib, sunitinib, temsirolimus and bevacizumab as single agents provided a survival advantage by inhibiting tumor growth and preventing weight loss in these mice. Blockade of human IL-6 with tocilizumab or ALD518 did not inhibit tumor growth and weight loss still occurred, demonstrating the possibility of other pathways involved in the cachexia mechanism. In addition, the SOC data for sunitinib showed significant tumor growth inhibition, but no regression at the study end point. This correlated with the clinical profile, where sunitinib could not prevent metastasis and the patient eventually progressed. The TumorGraft model was molecularly characterized and mutation analysis revealed a KIT V530I mutation. This mutation has been reported in extra-abdominal aggressive fibromatosis (desmoid tumor) and acute myeloid leukemia patients that have responded to c-kit targeting therapies, such as imatanib and dasatinib. Further characterization of this TumorGraft is ongoing to include additional bioinformatics using gene expression and mutation pathway analyses. In summary, we have continued development of this cachexia model using the Champions TumorGraft platform, along with molecular evaluation, to produce a robust and well-characterized approach for cachexia investigation which can also be applied to other areas of drug development and cancer research.

Citation Format: Nathan Anderson, Tin Khor, Andrew Feldhaus, Andreya Gatling, Katie Olson, John Latham, David Sidransky, Elizabeth M. Bruckheimer. Development of a spontaneous in vivo cachexia model using the Champions TumorGraft™ platform. [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 1217. doi:10.1158/1538-7445.AM2014-1217
Abstract 122: 15-lipoxygenase-1 regulation of HIF-1a and angiogenesis
Authors: Wu Yuanqing , Mao Fei , Zuo Xiangsheng , Moussalli Micheline J. , Elias Elias , Shureiqi Imad
Published: May 2015

Expression of 15-lipoxygenase-1(15-LOX-1) is down-regulated in colon and other major human cancer cells. 15-LOX-1 re-expression in cancer cells suppresses colonic tumorigenesis. We recently reported that targeted transgenic 15-LOX-1 expression in murine intestine suppresses colonic tumorigenesis. Nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia-inducible factor 1-alpha (HIF-1a) is a transcriptional master regulator that enhances cancer cell survival under hypoxia and the development of various metastatic mechanisms (e.g. upregulation of VEGF to promote angiogenesis). We therefore examined the effects of 15-LOX-1 expression restoration in colon cancer cells on cell survival, migration and invasion, angiogenesis, and HIF-1a expression and stability under hypoxia.

We found that 15-LOX-1 re-expression in colon cancer cells inhibited colon cancer cell survival, migration and invasion, VEGF expression, HUVEC cell tubular formation, and HIF-1a protein stability under hypoxia. These findings demonstrate that the loss of 15-LOX-1 significantly contribute to the ability of colon cancer cells to acquire metastasis promoting abilities by increasing a HIF-1 protein stability to up-regulate VEGF production of cancer cells and subsequently angiogenesis. Thus, therapeutic targeting 15-LOX-1 ubiquitous loss in cancer cells has the potential to be used to suppress tumor metastases.

Citation Format: Yuanqing Wu, Fei Mao, Xiangsheng Zuo, Micheline J. Moussalli, Elias Elias, Imad Shureiqi. 15-lipoxygenase-1 regulation of HIF-1a and angiogenesis. [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 122. doi:10.1158/1538-7445.AM2014-122
Abstract 1226: Breast and ovarian cancer PDXs show excellent conservation of original features but are genetically plastic
Authors: Theillet Charles G. , Manoir Stanislas du , Orsetti Beatrice
Published: May 2015

Patient derived xenografts (PDXs) are increasingly appreciated models in cancer research and preclinical testing. We have established a collection of 20 breast and 20 ovarian cancer PDXs and investigated their biological and clinical characteristics, as well as their genetic stability. Take rates were distinctly lower with breast (16%) compared to ovarian cancers (85%). The majority of our breast PDX originated from triple negative (14/20), grade 3 tumors (18/20) and breast cancers with reduced recurrence free survival. Interestingly, we stabilized 1 ER-/HER2+ and 5 ER+ PDX. Overall we showed that breast tumors that gave rise to a PDX (take) showed significantly different expression profiles compared to tumors which did not take. Ovarian PDX originated all from high grade cancers, but comprised all major ovarian cancer histotypes (mucinous, clear cell, carcinosarcoma and serous carcinoma).

Breast and ovarian PDXs faithfully recapitulated the histological features of their tumors of origin. The excellent conservation of original transcriptome characteristics was a further proof of the phenotypic stability shown by our PDX models. Noticeably, all grafts from ER+ cancers expressed ER at the RNA and protein levels. Array-CGH analysis revealed that PDXs presented a high degree of conservation of the Copy Number Change (CNC) profiles of the tumors they stemmed from and profiles from PDXs established from the same original tumor were highly overlapping. We determined that CNC representing up to 6% of the genome corresponded to oligoclonal events present in the tumor of origin that got gradually lost in the PDXs along passages. Furthermore, we noted that a minority of the PDXs presented a fraction of de novo occurring events representing up to 10% of the genome, suggesting limited levels of ongoing genetic instability.

In conclusion, our data show that breast and ovarian cancer PDX are excellent reproductions of the tumors of origin but remain genetically dynamic elements susceptible to drift during sequential passages.

Citation Format: Charles G. Theillet, Stanislas du Manoir, Beatrice Orsetti. Breast and ovarian cancer PDXs show excellent conservation of original features but are genetically plastic. [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 1226. doi:10.1158/1538-7445.AM2014-1226
Abstract 1231: In vitro anticancer activity of imidazo-acridinone (HKH40A) in panels of human patient-derived tumor xenografts (PDXs) and human leukemia and lymphoma cell lines
Authors: Fiebig Heinz H. , Maier Armin , Kelter Gerhard , Vuaroqueaux Vincent , Hariprakasha Humcha K. , Michejda Christopher
Published: May 2015

HKH40A is a synthetic, DNA-intercalating (Kosakowska-Cholody et al., 2009) anticancer agent consisting of an imidazo-acridinone linked to a 1,8-naphthalimide moiety via a 1,4-dipropylpiperazine linker. It displayed strong inhibitory activity in the NCI-60 tumor cell line panel in vitro with an IC50 of about 1 nM in selected tumor cell lines (Hariprakasha et al. 2007). In addition, anticancer activity was observed in hepatoma models both in vitro and in vivo. We have investigated the in vitro anticancer activity of HKH40A in 3D cultures, using panels of 206 PDXs and 11 human leukemia and lymphoma cell lines. The 3D clonogenic assay was performed in a 96 well format using a metabolic readout as an equivalent for colony formation. PDXs and hematologic cell lines were propagated in immunedeficient mice.

HKH40A proved to be very potent with a mean IC50 of 6 nM and a mean IC70 of 12 nM. Overall, the hematological cell lines were more sensitive than the solid tumor models. Selective activity (definition: IC50 < 1/3 of mean IC50) was seen in 4 out of 6 acute myeloid leukaemias (AMLs), in 1 out of 1 chronic lymphocytic leukemia (CLL) and in 1 out of 3 non-Hodgkin lymphomas (NHLs). Among the solid tumors, pleural mesotheliomas were most sensitive followed by soft tissue sarcomas and anal cancer. Selective activity was observed in a subset of 8 out of 30 colon cancer models, which presented IC50 values < 1/3 of the mean IC50 of all tumors. The drug sensitivity data will be correlated to mutational and transcriptomic (Whole Exome Sequencing and Affymetrix HG-U133 plus 2.0 array profiles) characteristics of the PDXs to identify predictive biomarkers of drug response and resistance.

Overall, HKH40A is a novel imidazo-acridinone with high in vitro activity mainly in AMLs and NHLs as well as in pleural mesotheliomas, soft tissue sarcomas and in a subset of colon cancer models. The identification of predictive biomarkers is ongoing.

*Deceased during an NCI retreat

Citation Format: Heinz H. Fiebig, Armin Maier, Gerhard Kelter, Vincent Vuaroqueaux, Humcha K. Hariprakasha, Christopher Michejda. In vitro anticancer activity of imidazo-acridinone (HKH40A) in panels of human patient-derived tumor xenografts (PDXs) and human leukemia and lymphoma cell lines. [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 1231. doi:10.1158/1538-7445.AM2014-1231
Abstract 1232: Antiproliferative, antioxidant and antiapoptotic effect of rhamnetin in human prostate cancer cells
Authors: Oak Christine , Bhaskaran Natarajan , Gupta Sanjay , Shukla Sanjeev
Published: May 2015

Persistent oxidative stress due to generation of reactive oxygen species (ROS) has been associated with malignancy and cancer progression. Using a panel of human prostate cancer cells, we observed increased production of intracellular ROS, compared to normal human prostate epithelial cells, accompanied by elevated expression of antioxidant enzymes viz. superoxide dismutase, catalase, peroxiredoxin-1 and thioredoxin that may be a likely mechanism to endure increased oxidative stress. There is a need for novel antioxidant agent that can effectively suppress the generation of increased ROS in cancer cells. Rhamnetin (3,5,3,4-tetrahydroxy-7-methoxyflavone), remains highest on oxygen radical absorbance capacity scale, however its anti-proliferative activities has not been assessed. Rhamnetin is an O-methylated flavonol, abundantly present in cloves, sweet annie, annual wormwood, green vegetables and orchard grass. Previous studies (Shukla et al. Proc. Amer. Assoc. Cancer Res. 102: 5576, 2011) using androgen-responsive human prostate cancer LNCaP and androgen-refractory PC-3 cells, we have demonstrated that rhamnetin possess anticancer activity, induces apoptosis, and inhibits cancer cell growth. In the present study we assessed the antioxidant and anti-proliferative ability of rhmanetin in prostate cancer. Treatment of LNCaP and PC-3 cells with 100μM paraquat, an oxidative stress-inducing agent, induced cellular proliferation in both these cells. Treatment of cells with 5-80μM rhamnetin significantly decreased paraquat-induced ROS production and inhibited cell proliferation in dose-dependent manner. Similar effects were noted with the addition of recombinant Klotho protein to these cells, which is known to offer resistance to oxidative stress at the cellular level. Rhamnetin treatment also increased Klotho protein levels in both cell lines, along with increase in the expression of p53 and p21/waf1. Furthermore, rhamnetin treatment resulted in the increased expression of caspase-3, caspase-9 causing induction of apoptosis, which may be one of the protective mechanism(s) to decrease cell proliferation and enhance resistance to oxidative stress in cancer cells. Taken together, these results demonstrate that rhamnetin acts as a potent antioxidant agent, reduces intracellular ROS levels, increases Klotho protein expression, induces apoptosis in prostate cancer cells, which may have a significant role in the inhibition of prostate cancer progression.

Citation Format: Christine Oak, Natarajan Bhaskaran, Sanjay Gupta, Sanjeev Shukla. Antiproliferative, antioxidant and antiapoptotic effect of rhamnetin in human prostate cancer cells. [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 1232. doi:10.1158/1538-7445.AM2014-1232