bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4949: Combined immunochemotherapy for poor prognosis breast cancer.
Authors: Wasiuk Anna , Rugo Hope , Coussens Lisa M.
Published: August 2014

Using preclinical mouse models of luminal B-type mammary adenocarcinoma (e.g., MMTV-PyMT mice), we, and others have reported that immune microenvironments of mammary tumors can be therapeutically manipulated to enhance responses to chemotherapy (CTX)(1). To determine the degree to which other subtypes of mammary carcinoma are similarly susceptible to immune reprogramming, we evaluated transgenic models of basal-like/triple negative (TN), e.g., C3(1)-Tag (2), and erbB2-amplified, e.g., Erbb2f/f/MMTV-Cre (3), mammary adenocarcinoma, with clinical/preclinical agents acting to either deplete or re-educate tumor-associated macrophages, in combination with taxol-based CTX, e.g., paclitaxel, nab-paclitaxel or eribulin mesylate. Our preliminary studies reveal similar complexity of immune cells infiltrating basal-like/TN tumors as what we previously reported for luminal B-type tumors where macrophages, as opposed to monocytes, are the predominate myeloid cells present. Significantly, mammary tumors in C3(1)-TAg mice exhibit improved responses to taxol-based CTX when given in combination with either colony stimulating factor-1 (CSF1) or CSF1 receptor (CSF1R) antagonists, as compared to either therapy alone, with no evident liver (ALT, AST, ALP) or kidney (Cre, BUN, CPK) toxicity. These studies reveal a synergistic benefit of combination macrophage-targeting immunotherapy with standard-of-care CTX regardless of mammary tumor subtype or form of taxol-based CTX. Thus, macrophage-targeting immunotherapy remains as an encouraging new neo-adjuvant strategy for slowing primary tumors and minimizing early metastasis. It remains to be determined if similar results will be realized with this class of agents in the metastatic setting.

1. DeNardo D, et al. Cancer Discov 1, 54-67 (2011)

2. Maroulakou I, et al., PNAS 91, 11236-11240 (1994)

3. Andrechek E, et al. PNAS 97, 3444-3449 (2000)

Support provided from the NCI/NIH (R01CA15531), the DoD (W81XWH-11-1-0702) and the Susan G Komen Foundation (KG110560, KG111084).

Citation Format: Anna Wasiuk, Hope Rugo, Lisa M. Coussens. Combined immunochemotherapy for poor prognosis breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2013-4949
Abstract 496: Inflammatory responses with infiltrating DC and functional cytotoxic T lymphocytes in the dermal vaccination sites of GVAX/ipilimumab treated prostate cancer patients.
Authors: Santegoets Saskia J. , van Mens Leonieke JJ , Beliën Jeroen AM , Stam Anita GM , Lougheed Sinead M. , Scheper Rik J. , Samiei Shabnam , Gall Helen , Jooss Karin , Cuillerot Jean-Marie , Sacks Natalie , Hege Kirsten , Gerritsen Winald R. , van den Eertwegh Alfons JM , de Gruijl Tanja D.
Published: August 2014

We have been studying the safety and clinical efficacy of the Prostate GVAX vaccine (consisting of two allogeneic GM-CSF secreting tumor cell lines: LNCaP and PC3) and the CTLA-4 blocking antibody ipilimumab in prostate cancer patients in a phase I/II dose escalation/expansion trial. To assess immune reactivity against the GVAX vaccine, immune infiltrate analysis of the vaccination sites was performed. Punch biopsies of the vaccination site were taken 48 hours after the 1st, 5th, and 11th injections and analyzed by immunohistochemical staining. Injection of the GVAX vaccine evoked striking local reactions that were characterized by local erythema, induration and swelling in all patients. Analysis of the vaccination site biopsies revealed that the GVAX/ipilimumab combination resulted in an inflammatory response in the skin with intradermal infiltrates of CD1a+ dendritic cells (DC), DC-SIGN+/CD68+ immature DC/macrophages, CD14+ monocyte-like cells, eosinophils, and of CD4+, CD8+, and Granzyme-B+ T lymphocytes. Strikingly, CD56+ NK cells were completely absent. The inflammatory response significantly increased over the course of treatment and resulted in accelerated clearance of the vaccine in vivo. Moreover, analysis revealed that the PC3 vaccine was more immunogenic, and induced a more potent anti-vaccine T cell response. The latter was confirmed in an in vitro cytotoxicity assay. Upon multiple vaccinations T cell reactivity to PC3 was significantly enhanced in the PC3 but not in the LNCaP vaccination sites, whereas reactivity to LNCaP was not enhanced in either vaccination site. We conclude that the GVAX/ipilimumab combination elicits a local immune infiltrate that increases over time, consistent with an induced CD8+ effector T cell mediated cytolytic response, only in vaccination sites of the PC3 component of the vaccine.

Citation Format: Saskia J. Santegoets, Leonieke JJ van Mens, Jeroen AM Beliën, Anita GM Stam, Sinead M. Lougheed, Rik J. Scheper, Shabnam Samiei, Helen Gall, Karin Jooss, Jean-Marie Cuillerot, Natalie Sacks, Kirsten Hege, Winald R. Gerritsen, Alfons JM van den Eertwegh, Tanja D. de Gruijl. Inflammatory responses with infiltrating DC and functional cytotoxic T lymphocytes in the dermal vaccination sites of GVAX/ipilimumab treated prostate cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2013-496
Abstract 5001: Cachexia induced different tumor microenvironment.
Authors: Matsuyama Tatsuzo
Published: August 2014

Back ground and aim

Cancer cachexia affects up to 80% of patients with advanced cancers and accounts for nearly 30% of cancer-related deaths.Cachexia is pointed out of loss of fat mass, wasting of skeletal muscle, and cardiac atrophy. The mechanism by which the degree of cachexia is induced by the different local cancer environment has not been studied. In this study, we investigated the mechanism of cachexia in the different local tcarcinoma such as between subcutaneous tumor model and intraperitoneal tumor model.

Materials and Methods

Cachexia was induced by subcutaneous grafting and intraperitoneal administration using colon cancer cells, (colon 26cells 1 × 10 ˆ 6/ 100μL each) in7-week-old BALB/c mice. Two weeks after the tumor-bearing, we measured adipose tissue (around epididymis), gastrocnemius muscle, heart, and cytokines in serum.

Result

The body weight, heart, fat, muscle weight of each mice was decreased gradually as of significant difference in subcutaneous tumor model, and intraperitoneal tumor model compared with normal mice . We have a significant difference in IL-1a, IL-4, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, G-CSF, IFN-γ, KC, MCP-1, MIP I-1a in the serum cytokine of tumor models compared with normal models. IL-12(p40), IL-13, G-CSF, KC has significant differences in subcutaneous tumor model compared with intraperitoneal tumor model.

Conclusion

In addition to the skeletal muscle and fat, in the state of cachexia it has caused in smooth muscle. And these change of cachexia is induced by not only one cytokine but several cytokines. Other important result is that cachexia is induced different degree by tumor origin.

Citation Format: Tatsuzo Matsuyama. Cachexia induced different tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5001. doi:10.1158/1538-7445.AM2013-5001
Abstract 5005: Deletion of bone morphogenetic protein receptor 1A impairs mammary gland development and tumor formation.
Authors: Owens Philip , Bobbitt Charli R. , Pickup Michael W. , Gorska Agnieszka E. , Novitskiy Sergey V. , Moses Harold L.
Published: August 2014

Bone Morphogenetic Proteins (BMPs) are secreted cytokines/growth factors belonging to the Transforming Growth Factor β (TGFβ) superfamily. BMP ligands have recently been shown to be overexpressed in human breast cancers. Normal breast and cancers display active BMP signaling as indicated by phosphorylated Smads 1, 5 and 8. BMP signaling is a known morphogen, responsible for differentiation of many cell types. This led us to hypothesize that BMP signaling may be required for development of the mammary gland and tumors. We first utilized the mammary epithelial specific Cre recombinase driven by the MMTV promoter to remove conditionally floxed BMPR1a alleles. We found that mice lacking BMPR1a (cKO) had significant reduction in epithelial branching morphogenesis within the mammary gland. We further analyzed BMPR1a loss in lactating mice using WAP promoter driven Cre, which demonstrated a loss of milk production. We next combined the MMTV.PyVmT oncogene with mice lacking BMPR1a and found this deletion resulted in delayed tumor onset and extended survival significantly (p-value <.001). Furthering these studies, we investigated the morphology of BMPR1a deleted tumors and found a striking loss of luminal characteristics combined with a stromal desmoplasia. Immunofluorescence staining revealed that cKO tumors co-expressed cytokeratins and mesenchymal cell markers such as Vimentin. This indicated that epithelial-to-mesenchymal (EMT) like changes were occurring upon BMPR1a deletion. We performed a DNA microarray on these tumors and found unique changes, which supported a loss of mammary gland differentiation and resulted in EMT-like activation. We next examined human breast tumor cell lines that previously reported the molecular signature response to BMP stimulation and compared them to primary human mammary epithelial cells (HMEC). Comparing signatures, we found that BMP4 stimulation induced unique genes in HMECs compared to tumor cells. Interestingly, transcriptional targets that were absent from tumors and found in normal HMECs included differentiation related genes. These results suggest that BMP signaling is critically deregulated in tumors. In conclusion, we found that loss of the BMP pathway restricts mammary gland development and impairs tumor formation and progression.

Citation Format: Philip Owens, Charli R. Bobbitt, Michael W. Pickup, Agnieszka E. Gorska, Sergey V. Novitskiy, Harold L. Moses. Deletion of bone morphogenetic protein receptor 1A impairs mammary gland development and tumor formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5005. doi:10.1158/1538-7445.AM2013-5005
Abstract 5010: Characterization of a novel cell type in human renal proximal tubules with connection to renal cell carcinoma development.
Authors: Hansson Jennifer , Hultenby Kjell , Cramnert Catharina , Lindgren David , Axelson Håkan , Johansson Martin E.
Published: August 2014

The proximal tubular segments of the kidney are extremely energy demanding, rendering this epithelium exceptionally sensitive to ischemic insults. Acute renal injury is a potentially life threatening condition, where the tubular cells detach and die. However, the tubules of injured kidneys have a substantial regenerative ability, and epithelial continuity, can with proper supportive care be restored. The cellular source of regeneration has been debated and until now the proximal tubules has been considered a completely homogenous cellular epithelium.

Using an assay based on aldehyde dehydrogenase (ALDH) activity followed by FACS, we have recently discovered a cell with progenitor cell characteristics scattered within the proximal tubules of the kidney. These novel proximal tubule rare cells (PTRCs) expressed stem cell markers CD133 and CD24, as well as intermediate filaments not normally seen in PTs. The gene expression profile of these cells shows a striking resemblance to that of papillary renal cell carcinoma (pRCC) and the gene signatures are also associated with a worse prognosis in clear cell renal carcinoma. Additionally, cells positive for PTRC-markers were seen in regenerating tubules after acute kidney injury. We have characterized these cells further at an ultra structural level, using electron microscopy. The cells were small, flask shaped and had a more densely packed chromatin pattern and drastically fewer mitochondria than the surrounding proximal tubular cells, indicating a different metabolic state. We suggest that these cells, in contrast to the surrounding highly oxygen dependent proximal tubular cells, constitute renal reserve cell pool capable of surviving ischemic renal injury with a capacity to spearhead subsequent tubular repopulation. We suggest that the increased probability for survival of these cells is due to their low mitochondrial content, structural robustness and anti-apoptotic signaling. Also, due to the considerable transcriptional similarities between pRCC, renal adenomas and the PTRCs we hypothesize that pRCC may arise through multistep oncogenic transformation of PTRCs.

Citation Format: Jennifer Hansson, Kjell Hultenby, Catharina Cramnert, David Lindgren, Håkan Axelson, Martin E. Johansson. Characterization of a novel cell type in human renal proximal tubules with connection to renal cell carcinoma development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2013-5010
Abstract 5016: The human sub-ependymal zone harbors glioblastoma precursors and represents a distinct therapeutic target.
Authors: Piccirillo Sara Grazia Maria , Spiteri Inma , Sottoriva Andrea , Touloumis Anestis , Ber Suzan , Price Stephen J. , Heywood Richard , Francis Nicola-Jane , Collins Vincent P. , Venkitaraman Ashok R. , Curtis Christina , Marioni John C. , Tavare’ Simon , Watts Colin
Published: August 2014

Glioblastoma (GB), the most common and aggressive adult brain tumor, is characterized by phenotypic diversity and ultimately treatment failure. Using fluorescence-guided sampling we integrated copy number aberration, gene expression and molecular clock data to show that the sub-ependymal zone (SEZ) contains precursor cells of the corresponding mass. The genetically distinct tumor margin contains non self-renewing cells that retain the potential to regenerate the tumor. Functional assays confirmed that SEZ and marginal disease were less clinically aggressive compared to the mass compartment in the same tumor. However, resistance to supra-maximal chemotherapy doses and differential patterns of drug response were observed between compartments. These data provide novel insights into the basis of clinical diversity, poor treatment response and emergence of resistant disease in GB patients.

Citation Format: Sara Grazia Maria Piccirillo, Inma Spiteri, Andrea Sottoriva, Anestis Touloumis, Suzan Ber, Stephen J. Price, Richard Heywood, Nicola-Jane Francis, Vincent P. Collins, Ashok R. Venkitaraman, Christina Curtis, John C. Marioni, Simon Tavare’, Colin Watts. The human sub-ependymal zone harbors glioblastoma precursors and represents a distinct therapeutic target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5016. doi:10.1158/1538-7445.AM2013-5016
Abstract 5031: R-spondin potentiates Wnt/β-catenin signaling in Ewing sarcoma cells.
Authors: Pedersen Elisabeth A. , Scannell Christopher A. , Lawlor Elizabeth R.
Published: August 2014

Proper regulation of Wnt signaling is highly context-dependent and critical for growth and cellular maintenance. It is well-established that dysregulation of the canonical Wnt/β-catenin pathway contributes to the development and progression of numerous malignancies. Despite this, there has been little evidence to support a role for canonical Wnt signaling in Ewing sarcoma (ES) pathogenesis. Recent studies have shown that when members of the leucine-rich G-protein coupled receptor (LGR) family, namely LGR4 and LGR5, are bound by their ligands, R-spondins, they potentiate Wnt/β-catenin activity. Importantly, recent studies from our lab have shown that some ES tumors and cell lines express high levels of LGR5. In addition, R-spondin is highly expressed in the developing bone, which is the primary site of ES in children. Since ES cells do not exhibit canonical Wnt/β-catenin activity in standard culture conditions, we hypothesized that cues from the tumor microenvironment may be required for activation of this pathway. In particular, we hypothesized that R-spondins would potentiate Wnt activity in LGR-expressing ES cells. To test this hypothesis, ES cells were cultured in the presence of WNT3A and/or R-spondin 2 (Rspo2). Immunocytochemistry was used to determine β-catenin nuclear localization, and Wnt transcriptional activity was determined by flow cytometry and luciferase activity using cell lines transduced with lentiviral 7xTCF-GFP and 7xTCF-luciferase reporters, respectively. qRT-PCR was used to confirm transcription of the Wnt target gene AXIN2. Our results show that exogenous WNT3A induces β-catenin nuclear localization in ES cells and is associated with induction of TCF-mediated transcriptional activity and AXIN2 expression. Furthermore, Wnt/β-catenin activation is potentiated in the presence of Rspo2, and ES cells lines with the highest expression of LGRs show the strongest potentiation. Interestingly, however, exogenous WNT3A and Rspo2 did not induce Wnt activity in all cells. Together these data confirm that in the presence of exogenous ligand, Wnt/β-catenin signaling can be robustly activated in a subpopulation of ES cells and that signaling is potently enhanced in the presence of Rspo2 and LGRs. To our knowledge, this is the first evidence of R-spondins influencing Wnt activity in a non-epithelial cancer. These studies support the concept that R-spondin expression in the tumor microenvironment may contribute to ES pathogenesis by modulating Wnt/β-catenin signaling.

Citation Format: Elisabeth A. Pedersen, Christopher A. Scannell, Elizabeth R. Lawlor. R-spondin potentiates Wnt/β-catenin signaling in Ewing sarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5031. doi:10.1158/1538-7445.AM2013-5031
Abstract 5035: Stable transfection of the osteosarcoma cell line SaOs2, genetically transformed MSCs and osteoblasts with insulin-like growth factor 2 receptor.
Authors: Qu Huayi
Published: August 2014

Osteosarcoma (OS) is the most commonly seen primary malignant bone tumor in children and adolescents. With neo-adjuvant chemotherapy and other treatment regimens developed, event-free survival has increased to 70%, but the prognosis remains poor for those patients with unresectable or metastatic tumors or less responsive tumors. Insulin-like growth factor 2 (IGF2) and its receptor (IGF2R) are known to be over-expressed in several cancers including OS and have roles in regulating cellular growth and proliferation. Although IGF2R is thought to be a tumor suppressor, the research results in different experimental systems are variable.

Our recent studies have demonstrated that IGF2R is consistently over-expressed, relative to other known transmembrane receptors on the cell surface across, 4 standard and 10 patient-derived OS cell lines. To further elucidate the effects of IGF2R in OS, the OS cell line SaOS2 and two cell lines which were genetically transformed, as follows; mesenchymal stem cells (MSC) and osteoblasts (OB) with human telomerase reverse transcriptase and SV40Tag (MSC-TS and OB-TS) were transformed with IGF2R by lentiviral transfection. Drug resistant colonies were picked up 21 days after selection to obtain stably transfected SaOS2, MSC-TS, and OB-TS cell lines now named, SaI, MSC-TSI and OB-TSI, respectively. After cloning, the highest and the lowest IGF2R expression clones were selected from these different cell lines via real-time PCR and western blot for further analysis. The comparison between the lines and characterizations are underway. Initial results suggest introduction of IGF2R changes the phenotype by enhancing cellular motility. Further studies will be presented and include the following: 1) cell proliferation assays, 2) motility and migration assays, 3) subcutaneous tumorigenicity assays, 4) expression profiling, and 5) differentiation assays. In the context of osteosarcoma introduction of IGF2R enhances motility, which is unexpected given prior expectations based on the non-signaling nature of this receptor.

Citation Format: Huayi Qu. Stable transfection of the osteosarcoma cell line SaOs2, genetically transformed MSCs and osteoblasts with insulin-like growth factor 2 receptor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5035. doi:10.1158/1538-7445.AM2013-5035
Abstract 5057: Activation of stroma-derived matrix metalloproteinases (MMPs) in a model of gastric cancer.
Authors: Kandola Sandhir , Holmberg Christopher , Ghesquiere Bart , Impens Francis , Gevaert Kris , Kumar J. Dinesh , Cash Nicole , Hegyi Peter , Vlatkovic Nikolina , Wang Timothy , Dockray Graham , Varro Andrea
Published: August 2014

Gastric cancer is the second commonest cause of cancer-related death worldwide. Within these tumors, it is increasingly recognised that signaling between myofibroblasts, an important stromal cell type, and cancer cells results in proliferation, invasion and metastasis. However, little is known of the range of proteins secreted by stromal cells ie their secretome, or the contribution of proteases in shaping the extracellular milieu ie the cancer degradome.

Using SILAC labeling and combined fractional diagonal chromatography (COFRADIC) we identified and quantified differential abundance and appearance of neo-N-termini (ie cleavage products) in the secretomes of gastric cancer-associated myofibroblasts (CAMs) compared with myofibroblasts from adjacent tissue (ATMs). There was decreased abundance of many proteins in CAM secretomes, but a small sub-set exhibited increased abundance including matrix metalloproteinases (MMP) -1 and -3. Cancer-restricted proteolytic cleavages included cleavage in the prodomains of MMP-1, -2 and -3 consistent with activation. Western blot, enzyme activity and cancer cell migration assays verified significantly increased abundance of active MMP-1, -2 and -3 in CAM media.

To establish the in vivo relevance of these findings we utilized a model in which primary gastric CAMs stimulate growth of MKN45 gastric cancer cells in xenografts in nude mice. MMP activity was imaged by fluorescence molecular tomography (FMT) employing MMPSense750 FAST™, which produces a fluorescent signal following MMP cleavage. CAMs enhanced xenograft growth by 6-fold (mean ± SEM, 29.6±21.1 vs 196.8±32.2 mm3 n=12, p=0.004). Using an experimental design that achieved similar volumes for tumors containing MKN45 cells alone and those containing MKN45+CAM, FMT assessment of tumor-related MMP activity in vivo indicated a two-fold increase in CAM-MKN45 xenografts compared with MKN45 cells alone (0.9±0.4 vs 1.8±0.8 fmol/mm3).

Stromal cell secretomes therefore contribute to the remodelling of the cancer cell microenvironment by activation of MMPs with consequences for cell migration and tumor growth. This system has potential for in vivo screening of anti-cancer therapies directed at the inhibition of stroma-stimulated tumor growth.

Citation Format: Sandhir Kandola, Christopher Holmberg, Bart Ghesquiere, Francis Impens, Kris Gevaert, J. Dinesh Kumar, Nicole Cash, Peter Hegyi, Nikolina Vlatkovic, Timothy Wang, Graham Dockray, Andrea Varro. Activation of stroma-derived matrix metalloproteinases (MMPs) in a model of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5057. doi:10.1158/1538-7445.AM2013-5057
Abstract 5067: Human prostate epithelial cells secrete the tumor suppressor maspin as a soluble and exosome-associated protein .
Authors: Dean Ivory S. , Dzinic Sijana , Kaplun Alexander , Zou Yi , Mao Guangzhao , Sheng Shijie
Published: August 2014

Maspin, an epithelial-specific member of the serine protease inhibitor superfamily, inhibits tumor invasion and metastasis. Overall maspin expression is downregulated or lost in invasive and metastatic carcinomas. Maspin subcellular distribution among the nuclear, cytoplasmic and extracellular compartments is differentially regulated during tumor progression. Extracellular maspin has been shown to inhibit tumor cell invasion and motility in vitro. Also, recombinant maspin was shown to inhibit endothelial cell tube formation in vitro and tumor-angiogenesis in vivo. To date, the mechanisms governing maspin trafficking remain unclear. In the current study, we aim to investigate the mechanism and biological significance of maspin secretion. Normal and tumor cells known to secrete maspin, were treated with or without drugs targeting classical or non-classical secretory pathways. The CM were collected and analyzed for soluble and exosome-associated proteins. We also utilized atomic force microscopy to characterize the physical characteristics of exosomes. We found maspin as a soluble protein in the CM. Maspin secretion was not inhibited by the drug treatments. In fact, maspin secretion increased under drug treatment targeting the classical secretory pathway. Furthermore, maspin was detected in the exosomes. Under drug treatment, the overall increase in maspin secretion was accompanied by an increase in detection of maspin in the exosomes. Based on the drug treatment-induced changes in maspin's secretion patterns, we speculate that maspin is secreted via a non-classical pathway. In addition, we provide the first evidence that maspin is secreted inclusively as both a free and an exosome-encapsulated molecule in normal and tumor cells. The existence of extracellular maspin as a free and an exosome-associated molecule implies a dual role for maspin in the tumor microenvironment. These novel findings are currently under investigation since the tumor suppressive activity of exosome-associated maspin may be of particular significance in cell-cell communication, and tumor suppressive activity of free maspin may be associated with extracellular matrix remodeling.

Citation Format: Ivory S. Dean, Sijana Dzinic, Alexander Kaplun, Yi Zou, Guangzhao Mao, Shijie Sheng. Human prostate epithelial cells secrete the tumor suppressor maspin as a soluble and exosome-associated protein . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2013-5067
Abstract 5088: Targeting the mTORC2/HIF-2alpha/p53 pathway in clear cell renal cell carcinoma.
Authors: Selvarajah Jogitha , Moumen Abdeladim , Carroll Veronica A.
Published: August 2014

Inactivating mutations in the von Hippel Lindau (VHL) tumour suppressor gene leads to both hereditary and sporadic clear cell renal cell carcinoma (ccRCC). Loss of VHL function results in stabilisation of hypoxia inducible factors 1 and 2 (HIF-1α and HIF-2α) with HIF-2α critical for renal tumour progression, angiogenesis and resistance to radiotherapy and chemotherapy. Previous work in VHL defective RCC cell lines that express HIF-2α has shown that loss of HIF-2α, either with siRNA or by reintroduction of VHL, increases phosphorylation and stabilisation of p53 which restored sensitivity to radiation and chemotherapy. Furthermore, as recent evidence suggests that mTORC2 is required for HIF-2α protein synthesis in VHL defective cells, we investigated the effects of pharmacological inhibition of HIF-2α with the mTORC1/2 kinase inhibitor pp242 alone or in combination with DNA damaging agents. Phosphorylated p53 and total p53 levels were significantly elevated in VHL competent RCC cells lacking HIF-2α as compared with VHL defective cells, confirming a role for HIF-2α mediated inhibition of p53. We analysed the effects of the mTORC1/2 kinase inhibitor pp242 on HIF-2α protein levels by Western blot. pp242 inhibited phosphorylation of both mTOR and its substrate p70S6K and significantly reduced protein levels of HIF-2α as compared with the mTORC1 inhibitor rapamycin, which had no effect on HIF-2α. Since the DNA damaging agent camptothecin (CPT) is a potent inhibitor of HIF-1α we also determined its effects on HIF-2α expression. CPT reduced HIF-2α protein levels in VHL defective RCC cells and increased p53 stabilisation, which was significantly augmented in VHL competent cells following complete loss of HIF-2α. Consistent with its inhibition of the p53 pathway, HIF-2α prevented both spontaneous and CPT induced apoptosis of RCC cells as demonstrated by FACS analysis of cells in sub G1 and Western blot analysis of cleaved PARP. Taken together these results confirm that HIF-2α negatively regulates p53, confers resistance to DNA damage induced apoptosis in VHL defective RCC cell lines and that further studies investigating the pharmacological inhibition of HIF-2α mediated by mTORC1/2 kinase inhibitors alone or in combination with DNA damaging agents for the treatment of ccRCC are warranted.

Citation Format: Jogitha Selvarajah, Abdeladim Moumen, Veronica A. Carroll. Targeting the mTORC2/HIF-2alpha/p53 pathway in clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5088. doi:10.1158/1538-7445.AM2013-5088
Abstract 5101: Tanshinone II-A inhibits angiogenesis through down regulation of COX-2 in human colorectal cancer.
Authors: Li Qi
Published: August 2014

Tanshinone II-A Inhibits Angiogenesis through Down Regulation of COX-2 in Human Colorectal Cancer

Qi Li*, Li-Hong Zhou, Hua Sui, Yan Wang, Xuan Liu, Ning-Ning Liu, Pei-Hao Yin

Clinic Oncology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai China

*correspondence to Qi Li, Lzwf@hotmail.com

Angiogenesis plays a significant role in colorectal cancer (CRC). cyclooxygenase-2 (COX-2) is involved with multiple aspects of CRC angiogenesis. The aim was to investigate the inhibitory effects of Tan II-A(Tanshinone II-A, Tan II-A) on tumor growth in mice, as well as alteration of expression of COX-2 and VEGF in CRC. We established the mice xenograft model of C26 CRC cell line, and injected 0.5, 1, 2mg/kg of Tan II-A and 1mg/kg of 5-FU in different individuals respectively in vivo. Then, we assayed tumor weight and volume, evaluated microvascular density and expression of VEGF. COX-2 promoter and COX-2 plasmids were transfected into HCT-116 cells, followed by detection of COX-2 promoter activity by chemiluminescence, detection of COX-2 mRNA expression by fluorescence quantitative PCR. Taken together, the results showed Tan II-A could inhibit tumor growth and VEGF level in vivo. Cell experiment showed marked inhibitory effect of Tan II-A on COX-2 and VEGF of HCT-116 cell lines in a dose-dependent manner. That means Tan II-A can effectively inhibit tumor growth and angiogenesis of human colorectal cancer via inhibiting the expression level of COX-2 and VEGF.

Keywords: Tanshinone II-A - colorectal carcinoma - angiogenesis - cyclooxygenase 2 - vascular endothelial growth factor

Citation Format: Qi Li. Tanshinone II-A inhibits angiogenesis through down regulation of COX-2 in human colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5101. doi:10.1158/1538-7445.AM2013-5101
Abstract 5109: Integrated micro nanotechnology for the sensitive isolation of circulating tumor cells.
Authors: Yoon Hyeun Joong , Kim Tae Hyun , Zhang Zhuo , Ramnath Nithya , Wicha Max S. , Hayes Daniel F. , Simeone Diane M. , Nagrath Sunitha
Published: August 2014

Introduction and Objective: Circulating tumor cells (CTCs) have been identified in the art in peripheral blood from cancer patients and are likely the origin of metastatic disease. Isolation and capture of CTCs represent a potential alternative to invasive biopsies during diagnosis of disease. In the recent past, vVarious microfluidic- based CTC isolation technologieschips have been developed to capture, indentify, sort, and enumerate CTCs. but, they need to be more sensitive, reliable, and specific for CTC detection in early stage cancer patients. Here we present a microfluidic CTC-chip with functionalized nanomaterials to capture and culture CTCs.

Methods: Micro-sized structuresposts functionalized with anti-epithelial-adhesion-molecule antibodies (Anti-EpCAM) are is the common approachstructures to capture CTCs. Here we present a microfluidic device with functionalized planar structure to capture CTCs. The CTC-chipmicrofluidic device has a PDMS chamber and patterned gold postatterns . functionalized with graphene oxides as nanomaterial and anti-epithelial-cell-adhesion-molecule antibodies (Anti-EpCAM). To investigate the capture efficiency before running cancer patients’ samples, a low number of cells were spiked into 1 mL of whole blood. We stained the captured cells with cytokeratin, CD45, and DAPI to identify CTCs. For clinical studies, we searched for CTCs in 10 pancreatic cancer patients, 6 breast cancer patients, 4 early lung cancer patients, and 5 healthy donors.

Results: It is much easier to scan, count and grow captured cells in comparison with previous micro-sized post-based structure. CTCs were isolated by assembling Graphene oxide on the planar structures for enhanced sensitivity. is easy to functionalize the surface and sensitive to detect biomaterial. The functionalized graphene oxide has been used to selectively capture MCF-7 breast cancer cells with a capture rate higher than 8790 %. This new CTC-chip identified CTCs in the peripheral blood of patients with pancreatic, breast, and lung cancer in 20 of 20 samples (100%).

Conclusion: We present a sensitive CTC-chip using graphene oxides as nanomaterial to isolate, capture, identify, and characterize extremely rare CTCs. This CTC-chipIt is much easier to scan, count, and grow captured cells in comparison with previous micro-sized post-based structure. We believe this CTC-chip offers great potential for clinical research on cancer.

Citation Format: Hyeun Joong Yoon, Tae Hyun Kim, Zhuo Zhang, Nithya Ramnath, Max S. Wicha, Daniel F. Hayes, Diane M. Simeone, Sunitha Nagrath. Integrated micro nanotechnology for the sensitive isolation of circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5109. doi:10.1158/1538-7445.AM2013-5109
Abstract 5124: 12-Lipoxygenase regulation of prostate cancer progression.
Authors: Tovar Elizabeth , Tucker Stephanie , Honn Kenneth V.
Published: August 2014

The underlying mechanism by which elevated omega-6 fatty acids, like arachidonic acid, lead to increased prostate cancer (PCa) risk is unclear. However, it is known that the 12-Lipoxygenase (12-LOX) enzyme that metabolizes arachidonic acid, as well as the 12(S)-HETE metabolic end product together with its cognate receptor, 12-HETER1, are strongly associated with PCa. 12(S)-HETE affects many cancer phenotypes including cell migration and metastasis. It causes the disassembly of complex cellular anchors to the substrata called hemidesmosomes that are comprised of numerous proteins, including ITGB4, which we identified as an interacting partner of 12-LOX. ITGB4 also physically interacts with c-Met at the cell membrane in multiple cell lines, in a cell line and stimulus-specific manner. PCa metastases have increased levels of both 12-LOX and c-Met. Therefore we sought to determine if ITGB4, c-MET and 12-LOX comprise a signaling axis. Our findings revealed that HGF-c-Met-induced invasion could be attenuated by enzymatic inhibitors of 12LOX, but that addition of exogenous 12(S)-HETE could not overcome the block. Furthermore, the physical interaction between β4 and c-Met remained unaffected by enzymatic inhibitors in PCa cells. Likewise, LC-MS analyses, showed that HGF stimulation did not lead to increased 12(S)-HETE production, and knockdown of 12-HETER1, by shRNA had no effect on HGF-induced invasion. Therefore, we hypothesize that 12LOX has dual functions whereby its enzymatic activity leads to hemidesmosome dissociation and cell release from the substrata, and its capacity to modulate c-MET/ITGB4 signaling may be attributed to a scaffolding function.

Citation Format: Elizabeth Tovar, Stephanie Tucker, Kenneth V. Honn. 12-Lipoxygenase regulation of prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5124. doi:10.1158/1538-7445.AM2013-5124
Abstract 5130: Prostate circulating tumor cells metastasize to bone via E-selectin expressed on endothelial cells.
Authors: Gakhar Gunjan , YuLee Guang , Seandel Marco , Bander Neil , Nanus David
Published: August 2014

Abstract

Prostate cancer cells preferentially metastasize to bone. Studies show that the preferential adhesion is due to interactions between prostate tumor cells and E-selectin present on human bone marrow endothelial cells. In our study, we used a parallel flow chamber to identify if prostate circulating tumor cells (CTCs) metastasize using similar interactions. CTCs were investigated based on the fact that majority of prostate tumor cells exhibit prostate specific membrane antigen (PSMA) on their cell surface. Prostate cancer cells, MDA PCa 2b (MDA) were first labeled with anti-PSMA monoclonal antibody J591 conjugated with alexa fluor 488 (J591-488) that recognizes PSMA and is internalized following binding to PSMA. Anti-PSMA labeled MDA cells were then perfused over IL-β stimulated human umbilical vein endothelial cells (HUVECs) using parallel flow chamber under physiological blood flow. Both unlabeled and J591-488 labeled MDA cells showed similar rolling behavior on stimulated HUVECs. This suggests that prostate CTCs can be identified and their interactions with endothelial cells can be studied. Our study will potentially elucidate the mechanism involved in prostate cancer metastasis to bone.

Citation Format: Gunjan Gakhar, Guang YuLee, Marco Seandel, Neil Bander, David Nanus. Prostate circulating tumor cells metastasize to bone via E-selectin expressed on endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5130. doi:10.1158/1538-7445.AM2013-5130
Abstract 5146: Phosphositeplus® and protein modifications in cancer, a case study: profiling tyrosine phosphorylated sites in AML and CML.
Authors: Hornbeck Peter , Zhang Bin , Murray Beth , Skrzypek Elzbieta , Kornhauser Jon , Latham Vaughan
Published: August 2014

PhosphoSitePlus® (PSP) is an open, comprehensive, manually curated, and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses over 250,000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The results of thousands of mass spec experiments, many from various cancers, are aggregated and available for downloading from PSP. The cancers that are most widely represented in PSP include non-small cell lung cancer (NSCLC), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), breast cancer (BRC), and gastric (GAC) cancer. In order to determine if the sort of binary data in PSP can be useful in identifying sites that are differentially tyrosine phosphorylated between various cancers, 284 CML and 160 AML datasets were downloaded from PSP and analyzed. Differences in the expression of tyrosine phosphorylation were observed: sites that were elevated in AML included PAG1 Tyr317/359/417, WASP Tyr291, SHP-1 Tyr536, and E-Syt1 Tyr822. Sites that were elevated in CML included Bcr and Abl sites as expected, as well as DYRK1A Tyr145, VASP Tyr39, CD2AP Tyr88, SHIP-2 Tyr986/1135/1162. These meta-analyses demonstrate that PSP provides a rich source of easily accessible data that enables proteomic profiling of multiple cancers. See www.phosposite.org

Supported by NIH R44 CA126080, R43 GM65768, and R44 AA014848

Citation Format: Peter Hornbeck, Bin Zhang, Beth Murray, Elzbieta Skrzypek, Jon Kornhauser, Vaughan Latham. Phosphositeplus® and protein modifications in cancer, a case study: profiling tyrosine phosphorylated sites in AML and CML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5146. doi:10.1158/1538-7445.AM2013-5146
Abstract 5150: What is in control of DNA replication timing.
Authors: Gindin Yevgeniy , Meltzer Paul S. , Bilke Sven
Published: August 2014

The initiation of DNA replication at specific sites in the genome is known to be far from perfect, where known initiation sites may or may not fire in a given single cell during specific cell cycle. At the same time, from larger scale perspective, one finds that the timing of DNA replication is highly orchestrated, where specific regions in the genome are copied at specific, reproducible time points throughout S-phase. In an attempt to reconcile these two somewhat discordant observations, many researchers have assumed that a yet unknown, active regulatory mechanism in the cell is responsible for coordinating replication timing.

Here we present a mechanistic explanation for genome-wide replication timing patterns. Our model is purely stochastic, does not contain any active control components and reproduces existing replication timing data with extreme fidelity (correlation > 0.9, about the same as the correlation between technical replicates of the experiment). The main findings of our work are

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Existence of reproducible genome-wide replication patterns do not logically necessitate existence of a complex, active control system in the cell

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Integrative analysis of recently released ENCODE data identifies semi-local chromatin structure as the main determinant of replication timing

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Plasticity of replication timing across different cell types are a direct consequence of changes in the chromatin structure between the different cell differentiation states

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Replication timing is a “systems” phenomenon emerging from the collective action of many uncoordinated, stochastic replication initiation sites. Replication timing cannot be understood by investigating individual initiation sites: deleting up to 30% of high probability initiation sites does not significantly change replication timing

Citation Format: Yevgeniy Gindin, Paul S. Meltzer, Sven Bilke. What is in control of DNA replication timing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5150. doi:10.1158/1538-7445.AM2013-5150
Abstract 5166: Membrane phospholipid asymmetry mediates the selective packaging of protein factors in cancer microparticles.
Authors: Hou Songwang , Williams Carol , Zhao Ming
Published: August 2014

Objective: Growing evidence indicates that cancer cell-derived microparticles (MPs) play important regulatory roles on cellular and system levels. These activities are attributed to protein factors inside MPs. It has been known that contents in MPs are not a passive representation of the host cell, but a collection of factors packaged in a selective fashion. In the current study, we hypothesize that membrane phospholipid dynamics promotes the selective packaging of protein factors by modulating the electrostatic density inside MPs.

Methods: The dynamics of phospholipid species across the membrane bilayer, including phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositides (PIs) are characterized using specific molecular probes. We used exogenous probes, Duramycin and bovine lactadherin, to stain the presence of PE and PS, respectively, on the outer surface of MP membrane. Endogenously expressed probes include GFP-tagged C2 domain of lactadherin (LacC2-GFP), and GFP-tagged 2PH-PLCdelta, are used to assess the relative density of PS and PI, respectively, in the inner leaflet of MP membranes. We examined the distribution of small Rho GTPases, including RhoA, Cdc42 and Rac1, using GFP-labeled reporters. To further examine the electrostatic interactions, we used RhoA mutants with the polybasic amino acids mutated to Gln. Additionally, we constructed a GFP-PBR to validate the electrostatic-dependent packaging mechanism.

Results: PE is externalized to the outer surface of MPs. This is correlated with a greater density of negatively charged phospholipids, particularly PS, in the inner leaflet. The process is independent of apoptosis or the generation of apoptotic bodies. The redistribution of PE to the MP surface is correlated with an elevated presence of small GTPases in a PBR-dependent manner. Mutation of PBR abolished RhoA uptake in MPs. The incorporation of PBR to GFP significantly enhanced GFP localization in MPs. Conclusion: PE externalization to MP surface likely results in a greater density of negatively charged phospholipids, including PS, and to a lesser extent, PIs. An elevated density of anionic phospholipids provides an electrostatic attraction for membrane-associated proteins to MPs. This may provide a general mechanism for the selective sequestration of signal transduction factors in cancer cell-derived MPs.

Citation Format: Songwang Hou, Carol Williams, Ming Zhao. Membrane phospholipid asymmetry mediates the selective packaging of protein factors in cancer microparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5166. doi:10.1158/1538-7445.AM2013-5166