bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract SY37-01: Dissecting the canonical and noncanonical interactions of the BCL-2 family for therapeutic benefit
Authors: Walensky Loren D.
Published: May 2015

BCL-2 family proteins are key regulators of apoptotic and other signaling pathways in health and disease. Discrete amphipathic alpha-helices embedded within the protein infrastructure are essential interaction motifs that can inhibit or activate their physiologic targets. The canonical complex between a pro-apoptotic BCL-2 homology domain 3 (BH3) helix and a surface groove on anti-apoptotic members portrayed a molecular wrestling match between pro- and anti-apoptotic signals. If anti-apoptotic grooves are sufficient in number to bind and sequester the pro-apoptotic BH3 signals, cell survival prevails. In contrast, if the capacity to withstand pro-apoptotic assault is breached, cell death ensues. Not only did this protein interaction paradigm provide a mechanism for apoptotic regulation, but it also informed the development of drugs to reactivate cell death through targeted inhibition of anti-apoptotic BH3-binding pockets. Given the critical roles of amphipathic alpha-helices in mediating signal transduction, we advanced all-hydrocarbon stapling to refold bioactive alpha-helical peptides for use as research tools and prototype therapeutics. Depending on their amino acid composition and design, “stapled peptides” have proven to be structurally stable, protease resistant, and cell permeable agents capable of interrogating and modulating protein interactions in vitro and in vivo. Here, I present our latest applications of Stabilized Alpha-Helices of BCL-2 domains (SAHBs) to dissect the canonical and noncanonical interactions that directly regulate the functional activity of BCL-2 family proteins and their targets. Our findings broaden the opportunities to pharmacologically modulate cell death and other physiologic processes by revealing new protein terrain for therapeutic targeting.

Citation Format: Loren D. Walensky. Dissecting the canonical and noncanonical interactions of the BCL-2 family for therapeutic benefit. [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 SY37-01. doi:10.1158/1538-7445.AM2014-SY37-01
Abstract SY37-04: Bcl-2 family proteins as a paradigm for protein:protein interactions as chemotherapy targets
Authors: Andrews David W.
Published: May 2015

Many aspects of cell signaling are regulated by protein:protein interactions including growth factor receptor signal transduction pathways, transcriptional regulatory complexes and programmed cell death. The Bcl-2 family proteins regulate programmed cell death via a series of protein:protein interactions that occur in the cytoplasm and in membranes. These interactions are regulated by the amount of the proteins, their locations and by post-translational modifications. The multi-domain pro-apoptotic Bcl-2 family members Bax and Bak directly regulate mitochondrial outer membrane permeabilization (MOMP), an event widely accepted as committing most cells to apoptosis. MOMP results from an ordered series of steps beginning with activation of one or more Bcl-2 homology 3 proteins (BH3-proteins). Once activated, BH3-proteins bind to mitochondria, directly recruit and activate Bax and the constitutively membrane bound Bak. In some cases ‘activation’ involves releasing a previously activated Bax or Bak from inhibition by an anti-apoptotic protein of the Bcl-2 family. Oligomerization of integral membrane Bax and/or Bak culminates in membrane permeabilization. The embedded together model describes these interactions as a series of dynamic equilibria that change when the proteins interact with membranes. Because of their role regulating apoptosis in cancer cells and in response to chemotherapy, Bcl-2 family proteins are attractive targets for the development of small molecule inhibitors. The rational development of small molecules requires understanding in detail the molecular interactions that regulate activity. However, studying protein:protein interactions for membrane bound proteins is fraught with difficulties. I will describe some of the challenges of studying this family of proteins and the successes and surprises that resulted from our analyzing and perturbing their interactions for a variety of Bcl-2 family proteins in live cells. I will highlight recent data that suggests new avenues to targeting Bcl-2 family protein:protein interactions therapeutically.

Citation Format: David W. Andrews. Bcl-2 family proteins as a paradigm for protein:protein interactions as chemotherapy targets. [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 SY37-04. doi:10.1158/1538-7445.AM2014-SY37-04
Abstract SY40-04: Immune control of spontaneous tumor development
Authors: Li Ming
Published: May 2015

Long recognized as an evolutionarily ancient cell type to control development, tissue homeostasis, and immune responses to pathogens, macrophages are being rediscovered as regulators of several chronic disorders including cancer. Using a spontaneous mammary tumor model, here we show that tumor growth induces the differentiation and expansion of a unique subset of tumor-associated macrophages (TAMs) that are phenotypically and functionally distinct from mammary tissue macrophages (MTMs). Compared to MTMs, TAMs do not exhibit an “alternatively activated macrophage” phenotype, but express the adhesion molecule Vcam1, and have high proliferative capacity upon their differentiation from CCR2-expressing inflammatory monocytes. Importantly, TAM differentiation is dependent on the transcriptional regulator of the Notch signaling pathway, RBPJ; and TAM, but not MTM, depletion restores tumor-infiltrating cytotoxic T cell responses, and suppresses tumor growth. These findings demonstrate that TAMs represent a novel lineage of mononuclear phagocytes with critical regulatory functions in promoting T cell tolerance to tumors.

Citation Format: Ming Li. Immune control of spontaneous tumor development. [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 SY40-04. doi:10.1158/1538-7445.AM2014-SY40-04
Abstract SY42-03: Novel DNA repair inhibitors for radiosensitization and cancer therapy
Authors: Glazer Peter M.
Published: May 2015

There is growing interest in the development of molecules that can inhibit DNA repair or disrupt DNA damage response pathways for application to cancer therapy. Such inhibitors offer the potential to achieve synthetic lethality in the context of DNA repair deficient cancers and/or to sensitize tumors to chemotherapy and radiation. We have identified a series of structurally related small molecules that demonstrate increased toxicity to human cancer cells deficient in BRCA2, PTEN, ATM, and the Fanconi pathway. These compounds are not PARP inhibitors but rather impact DNA damage response pathways at upstream steps, causing impairment of both homology-dependent DNA repair (HDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks. The molecules also reduce the formation of DNA repair foci by several damage response factors, including 53BP1, BRCA1, and DNA-PK, following ionizing radiation treatment of cancer cells. Efforts to identify the molecular target of these compounds are underway.

In addition, we have found that a highly unusual lupus-derived, cell-penetrating autoantibody, 3E10, preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we find that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low dose doxorubicin. These findings identify a new antibody that may have clinical application in cancer, particularly in BRCA-related malignancies. Because lupus is associated with circulating autoantibodies reactive against host DNA, the work also suggests that lupus autoantibodies may be a valuable source of other potential therapeutic agents. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the unexpectedly low rates of breast and ovarian cancers observed in lupus patients.

Citation Format: Peter M. Glazer. Novel DNA repair inhibitors for radiosensitization and cancer therapy. [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 SY42-03. doi:10.1158/1538-7445.AM2014-SY42-03
Abstract A07: Breast tumor microenvironment shapes vascular response to endothelial HoxA5 expression
Authors: Northcott Josette , Cuevas Ileana , Layman Hans , Boudreau Nancy
Published: May 2015

Current anti-angiogenic therapy for the treatment of solid tumors is based on directed inhibition of growth factor signaling pathways essential for the development of new blood vessels. Despite evidence showing a positive correlation between angiogenesis and breast cancer progression, existing anti-angiogenic therapies have not proven effective at clinically managing breast tumors or prolonging patient survival. Several studies have shown that tissue microenvironment shapes local angiogenic responses and tumor progression - a finding that may partially explain the refractoriness of breast tumors to anti-angiogenic therapy. Data from our laboratory supports an anti-angiogenic role for the HoxA5 homeodomain containing transcription factor. Using the KRT14-HPV16 mouse model of skin cancer crossed with our tetracycline-regulated mouse line that expresses HoxA5 in the endothelium, we observed that HoxA5 reduced angiogenesis and slowed tumor progression in the skin. Thus, we hypothesized that constitutive endothelial expression of HoxA5 during mammary tumor development would prevent tumor growth and metastasis via modulation of the endothelial phenotype. Surprisingly, in the MMTV-PyMT mouse model of breast cancer, we observed that endothelial HoxA5 expression increased both primary tumor growth and lung metastasis. Mammary tumors from PyMT/HoxA5+ mice had larger areas of hypoxia and necrosis, as compared to primary tumors from control mice. Although we did not detect any change in intra-tumoral vascular staining (CD31+), we observed an increased number of large vessels and reduced vascular leakage in the primary tumors from PyMT/HoxA5+ mice. In contrast, subcutaneous injection of MMTV-PyMT mammary tumor cells into HoxA5 transgenic mice displayed significantly reduced tumor growth and decreased intra-tumoral vascularization, as compared to controls. This suggests that, unlike the anti-angiogenic properties of HoxA5 in the skin microenvironment, within the context of the mammary gland HoxA5 has vascular normalization effects. We conclude that the tissue microenvironment shapes the vascular response to anti-angiogenic agents and thereby controls tumor progression.

Citation Format: Josette Northcott, Ileana Cuevas, Hans Layman, Nancy Boudreau. Breast tumor microenvironment shapes vascular response to endothelial HoxA5 expression. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A07. doi:10.1158/1538-7445.CHTME14-A07
Abstract A43: Systemic tumor-stroma interactions are prognostic indicators of breast tumor invasiveness
Authors: Frankenberger Casey A. , Bainer Russell O. , Rabe Daniel C. , Saleh Sadiq , Park Morag , Gilad Yoav , Rosner Marsha R.
Published: May 2015

Cancer progression is critically dependent on specific molecular interactions between tumor cells and their microenvironment, but little is known about the global dynamics of this crosstalk within and across individuals. Here, we used an RNA sequencing approach with species-of-origin information in a xenograft mouse model to computationally resolve and compare tumor versus stromal transcriptional changes. Our study employed a triple-negative breast cancer (TNBC) model in which primary tumor invasiveness is controlled by the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP) allowing systemic observation of stromal response to invasive or noninvasive breast tumours at both proximal and distal sites.

Initially, we observed that expression levels of gene homologs in human tumor and mouse stroma are highly synchronized. Surprisingly, the set of genes most prognostic for metastasis-free survival in multiple clinical data sets were those whose mRNA expression was inversely correlated between tumor and stroma. These results were confirmed in an independent set of microarray data from tumor and stroma micro-dissected from primary human breast cancer patient tissue. Additionally, we found broad-based and invasion-specific gene expression changes in stromal tissues far from the primary lesion, with these results being confirmed in an independent set of samples. Of note, genes differentially expressed in the tumor-associated stroma were better classifiers of tumor phenotype than genes differentially expressed in the tumor alone. As well, stromal genes differentially expressed both locally and distally, are significant predictors of patient prognosis.

These findings demonstrate the potential for using patient tissue remote from the primary tumor as indicators of disease aggressiveness. Furthermore, these results suggest that stromal gene expression, acting to compensate for changes in the tumor, may have both prognostic and therapeutic implications.

Citation Format: Casey A. Frankenberger, Russell O. Bainer, Daniel C. Rabe, Sadiq Saleh, Morag Park, Yoav Gilad, Marsha R. Rosner. Systemic tumor-stroma interactions are prognostic indicators of breast tumor invasiveness. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A43. doi:10.1158/1538-7445.CHTME14-A43
Abstract A47: IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor
Authors: Padmanaban Guruprasath , Vadevoo Sri Murugan Poongkavithai , Chi Lianhua , Lee Byung-Heon
Published: May 2015

Targeted delivery of imaging agents and therapeutics to tumors would provide early detection and increased therapeutic efficacy against cancer. Using phage displayed-random peptide libraries, we have identified IL4RPep-1 (IL-4 receptor-binding peptide-1), CRKRLDRNC, that binds to IL-4 receptor (IL4R). IL4R is over-expressed on many types of cancer cells including lung cancer and breast cancer. Peptides have smaller size and in turn may exert better tissue penetration than bulky antibodies. IL4RPep-1 bound to H226 lung tumor cells that over-express IL4R, while little binding was observed in H460 lung tumor cells that express IL4R at low levels. When injected intravenously into nude mice bearing a subcutaneous H226 tumor, Cy7.5 near-infrared fluorescence dye-conjugated IL4RPep-1 selectively homed to tumor. IL4RPep-1-labeled liposomes containing doxorubicin showed more efficient anti-tumor growth activity than untargeted liposomes. In addition, IL4RPep-1-labeled, branched-type polyethyleneimine nanoparticles containing Bcl-xL siRNA more efficiently knocked down Bcl-xL expression and showed higher cytotoxic effect on cancer cells over-expressing IL4R than cancer cells expressing IL4R at low levels. These results suggest that IL4RPep-1 could be a useful probe for affinity-based imaging and drug delivery to tumor that over-expressing IL4R.

Citation Format: Guruprasath Padmanaban, Sri Murugan Poongkavithai Vadevoo, Lianhua Chi, Byung-Heon Lee. IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A47. doi:10.1158/1538-7445.CHTME14-A47
Abstract A71: TMPRSS4 proteolytically activates pro-uPA to induce cancer cell invasion
Authors: Min Hye-Jin , Kim Semi
Published: May 2015

The invasive nature of tumor cells is critical for cancer metastasis. Urokinase-type plasminogen activator (uPA), which is usually derived from stromal cells, is a well-known serine protease involved in invasion and metastasis. uPA is produced as an inactive single-chain protein (known as pro-uPA or sc-uPA) that is processed into the active disulfide-linked two-chain form of uPA by a proteolytic event. TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates colorectal tumor cell invasion, migration, and metastasis and increased TMPRSS4 expression is associated with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates uPA gene expression through c-Jun N-terminal kinase signaling activation to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. Here, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of invasion through the regulation of uPA. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion.

Citation Format: Hye-Jin Min, Semi Kim. TMPRSS4 proteolytically activates pro-uPA to induce cancer cell invasion. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A71. doi:10.1158/1538-7445.CHTME14-A71
Abstract A79: Mouse models of breast cancer identify oncogene-associated stroma subtypes
Authors: Saleh Sadiq M. , Savage Paul , Laferrière Julie , Cory Sean , Bertos Nicholas , Souleimanova Margarita , Zhao Hong , Zacksenhaus Eldad , Muller William , Hallett Michael , Park Morag
Published: May 2015

Breast Cancer is a highly heterogeneous disease involving complex crosstalk between tumor cells and their surrounding microenvironment (stroma). Previous work in our lab utilized laser capture microdissection and gene expression analysis to separate Breast Cancer stromal samples into distinct subtypes. Although this and other data demonstrate the importance of the stroma in tumor progression, the cause of these stromal changes are still poorly understood. We hypothesize that changes within stroma are related to individual oncogenic events within the tumor epithelium. Analyzing human data does not allow us to draw causative conclusions due to the presence of compounding factors such as genetic variance and living conditions. In an attempt to circumvent this challenge we used transgenic mouse models of breast cancer in the same strain of mice (FVB) with oncogenes driven by a mammary specific promoter (MMTV). By keeping these variables constant and only varying the oncogene: using either Neu-NDL-2-5, PyVMT or Wnt1 - allowed us to test the aforementioned hypothesis.

Class discovery and distinction confirmed that the epithelial and stromal samples from each mouse model were distinct, which validated our hypothesis. Pathway analysis of the genes that distinguish the stroma of the mouse models identifies genes linked to recruitment and activation of different stromal cells. Interestingly our findings corroborate with results from other labs using these mouse models.

Citation Format: Sadiq M. Saleh, Paul Savage, Julie Laferrière, Sean Cory, Nicholas Bertos, Margarita Souleimanova, Hong Zhao, Eldad Zacksenhaus, William Muller, Michael Hallett, Morag Park. Mouse models of breast cancer identify oncogene-associated stroma subtypes. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A79. doi:10.1158/1538-7445.CHTME14-A79
Abstract B02: Matrix stiffness regulates local metabolism of breast carcinoma cells
Authors: Burkel Brian , Ponik Suzanne , Morris Brett , Eliceiri Kevin , Keely Patricia
Published: May 2015

Dense breast tissue is one of the single largest risk factors for the development of breast cancer, and one of the primary proteins responsible for increased breast density is the core extracellular matrix (ECM) component, collagen. Similar to other ECM proteins, collagen plays a structural role underlying tissue organization and increased collagen deposition correlates to a stiffer ECM and cellular microenvironment. Interestingly, changes to the stiffness of the ECM or microenvironment have profound and poorly-understood effects on cell migration, cell proliferation, and cancer progression. Consistent with an expanding role of ECM stiffness in cell signaling, we report that increased matrix stiffness also affects cellular metabolism and respiration. Using specific pharmacological inhibitors and quantitative imaging modalities like fluorescence lifetime microscopy, we are able to show that changes in collagen stiffness can cause a metabolic shift towards a more glycolytic, Warburg-like equilibrium in breast carcinoma cells. Matrix stiffness regulates the expression of several metabolic enzymes, including PDHK-1, which is poised to regulate this shift.

Note: This abstract was not presented at the conference.

Citation Format: Brian Burkel, Suzanne Ponik, Brett Morris, Kevin Eliceiri, Patricia Keely. Matrix stiffness regulates local metabolism of breast carcinoma cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B02. doi:10.1158/1538-7445.CHTME14-B02
Abstract B07: Metabolic liabilities of human colon carcinoma spheroids are different compared to standard 2D cultures
Authors: Dranka Brian P. , Swain Pamela , Rogers George W. , Divakaruni Ajit S. , Neilson Andy , Ferrick David A.
Published: May 2015

The search for metabolic liabilities in tumor cells is typically performed in standard 2D cultures. This approach lacks the 3D context and heterogeneity of tumor tissue in vivo, which has significant impact on their metabolic behavior. One solution to modeling complex tumor cell interactions is to use cultured tumor spheroids. To assess bioenergetic function in individual spheroids, a modified, 96-well plate for the Seahorse Bioscience XFe96 Extracellular Flux Analyzer was created. Here, we demonstrate the capability of measuring mitochondrial oxygen consumption – a measure or mitochondrial respiration – and extracellular acidification – a measure of glycolytic flux – in individual HCT116 colon carcinoma spheroids. Spheroids were cultured using the hanging drop method for 4 days. Mitochondrial function was determined using the Cell Mito Stress Test which profiles 6 specific indices of mitochondrial activity. Cells grown in spheroids had a markedly increased spare respiratory capacity compared to cells in 2D culture. These data are consistent with a shift from the highly proliferative nature of 2D culture to a more physiological model where proliferation and differentiation is more balanced. We hypothesize that viewing metabolism holistically allows for a more complete view of cellular bioenergetic function and potential liabilities. To understand the metabolic potential of cells in the context of their basal metabolism, two new metrics for describing mitochondrial function were established. By stimulating maximal oxygen consumption by combined exposure to oligomycin and FCCP, and maximal extracellular acidification rate (ECAR) using rotenone, antimycin A, and oligomycin, the potential to switch metabolic pathways was determined. Metabolic Capacity Index was identified using the maximal responses in each direction, and Metabolic Flexibility Index was calculated as the angle of the line to identify cellular preference for OXPHOS or glycolysis. Interestingly, plastic-adherent HCT116 cells only exhibited potential to increase glycolysis, while HCT116 spheroids had a more balanced response. These data are consistent with the finding that spheroids have increased spare capacity. Implications of this work for future studies in cancer biology will be discussed.

Citation Format: Brian P. Dranka, Pamela Swain, George W. Rogers, Ajit S. Divakaruni, Andy Neilson, David A. Ferrick. Metabolic liabilities of human colon carcinoma spheroids are different compared to standard 2D cultures. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B07. doi:10.1158/1538-7445.CHTME14-B07
Abstract B31: Lung fibroblast enhances MMP-1 secretion when co-cultured with human lung cancer cells in the 4D lung cancer model
Authors: Mishra Dhruva Kumar , Thrall Michael J. , Kurie Jonathan M. , Kim Min P.
Published: May 2015

Tumor microenvironment plays an important role in cancer development, progression and metastasis. However, it is difficult to determine the effect of fibroblast on lung cancer cells in patients with lung cancer. Recently, we have developed a novel ex vivo 4D lung cancer model that mimic the biology of human lung cancer growth. In order to determine the viability of the normal lung fibroblast, Wi-38, when grown in the 4D model, we grew Wi-38, alone or with A549, in the epithelial space of the acellular matrix of the 4D model with media that was continuously perfused through the vascular space. We performed a lobectomy on days 4, 6, 8, 11 and 14 from the 4D model. We performed H&E of the lobectomy specimen of Wi-38 grown alone and immunohistochemistry for CK7 cells for the lobectomy specimen of A549 grown with Wi-38. Next, we grew either A549 or H1299 alone or with Wi-38 in the 4D model and collected circulating media. We measured the matrix metalloproteinase (MMP) profiles of the circulating media on days 3, 6, 9,12 and 15 using Millipore Luminex kit. We used student t-test to determine the significant difference of MMP levels between the two conditions. When lung fibroblast, Wi-38, was grown on the 4D model alone there was viable fibroblast on day 14; however, when Wi-38 was grown with A549, there was a significantly less number of fibroblast (CK7 negative cells) on day 14 (2±1%) compared to day 4 (50±9%, p < 0.0001). For both H1299 grown with Wi-38 and A549 grown with Wi-38, there were significantly higher levels of MMP-1 on day 3 and day 6 compared with cancer cells grown alone (p< 0.05). However, there was no significant difference in the level of MMP-10 for all days of the experiment. Our study shows that when Wi-38 fibroblast is cultured with A549, the amount of fibroblast decreases over time and A549 grows in the acellular matrix. The study shows that fibroblast enhances MMP-1 production but not MMP-10 production for both H1299 and A549 cells. Overall, this study shows that both human fibroblast and cancer cells can grow together on acellular matrix and fibroblast may modify tumor progression by production of MMPs.

Citation Format: Dhruva Kumar Mishra, Michael J. Thrall, Jonathan M. Kurie, Min P. Kim. Lung fibroblast enhances MMP-1 secretion when co-cultured with human lung cancer cells in the 4D lung cancer model. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B31. doi:10.1158/1538-7445.CHTME14-B31
Abstract B36: The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer
Authors: Roybal Jonathon D. , Peng David H. , Ungewiss Christin , Mandal Pijus K. , McMurray John S. , Byers Lauren A. , Gibbons Don L.
Published: May 2015

Lung cancer is the deadliest cancer in the United States due primarily to the development of metastatic disease, resulting in a 16% five year overall survival. Regrettably response rates are poor with the current modalities used to treat patients, which implies that a greater understanding of the biology of invasion and metastasis is indispensable. It is thought that the epithelial-mesenchymal transition (EMT) plays a crucial role in regulating invasion, migration, and metastasis. The microRNA-200 family (miR-200) is a key maintenance factor of the epithelial phenotype by regulating EMT and metastatic potential, although the mechanistic details of this regulation are incompletely understood. Recent research has demonstrated that miRNAs are capable of regulating the target mRNAs that coordinate intracellular signaling pathways. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown through Reverse Phase Protein Array (RPPA) profiling that high levels of miR-200 in poorly metastatic lung tumor cells can suppress cancer-promoting signaling molecules, such as Signal Transducer and Activator of Transcription 3 (STAT3). In contrast, highly metastatic lung tumor cells with low miR-200 expression have STAT3 activation and increased migratory and invasive properties. Utilizing pharmacologic approaches to inhibit STAT3 in highly metastatic lung tumor cells decreased their ability to migrate and invade. Our data suggests that the STAT3 pathway is under the control of miR-200 and inhibition in metastatic lung cancer could provide benefit to lung cancer patients as a potential treatment option.

Citation Format: Jonathon D. Roybal, David H. Peng, Christin Ungewiss, Pijus K. Mandal, John S. McMurray, Lauren A. Byers, Don L. Gibbons. The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B36. doi:10.1158/1538-7445.CHTME14-B36
Abstract B70: Targeting the Tie2/angiopoietin signaling pathway in glioblastoma
Authors: Plate Karl H. , Scholz Alexander , Harter Patrick , Mittelbronn Michel , Slyke Paul van , Dumont Dan , Reiss Yvonne
Published: May 2015

Angiogenesis inhibitors have evolved in the past decade as one of the most promising biology based therapeutic strategies. Promising pre-clinical studies (published in 1993 and 1994) provided proof of principle that blocking of VEGF dramatically inhibits tumor growth. These observations led to the successful development of inhibitors for VEGF and VEGF receptors by various pharmaceutical companies. Bevacizumab, a monoclonal antibody neutralizing VEGF, was the first-in class drug to achieve FDA approval for the treatment of colorectal carcinoma in 2004 and later on in other cancer types, including glioblastoma. Anti-VEGF therapy led to an increase in progression free survival in recurrent and primary GBM, but its role in first-line treatment is less clear. Importantly, several preclinical studies suggested that resistance to anti-angiogenic therapy might evolve in the course of the treatment due to an infiltration of specially polarized myeloid cells.

In order to define new therapeutic options for anti-angiogenic therapy we investigated the Tie2/Angiopoietin (Angpt) signaling pathway in human and murine glioblastomas. In more than 200 human brain tumor specimens investigated, Angpt2 protein expression was found to be up-regulated in tumor endothelial cells whereas it was absent in the normal brain. Further, Angpt2 expression levels correlated with WHO grade as well as the number of infiltrating monocytes/macrophages. Notably, transgenic mice with endothelial cell-specific expression of Angpt2 showed a significant higher number of infiltrating myeloid cells, thus corroborating our findings in human GBM. These findings suggest, that Angpt signaling links inflammation and angiogenesis in GBM and may thus provides a therapeutic target.

In a GL261 orthotopic syngeneic glioma model, interference with Angpt signaling with pharmacologic agents and peptide-based blockers of Angpt2, lead to a significant increase in survival, increased pericyte coverage and altered myeloid cell/T-cell composition. Combining anti-Angpt with anti-VEGF based therapies showed synergistic effects. These findings argue for targeting the Tie2/Angpt signalling pathways in human GBM, either alone or in combination with anti-VEGF therapies.

Citation Format: Karl H. Plate, Alexander Scholz, Patrick Harter, Michel Mittelbronn, Paul van Slyke, Dan Dumont, Yvonne Reiss. Targeting the Tie2/angiopoietin signaling pathway in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B70. doi:10.1158/1538-7445.CHTME14-B70
Abstract B71: Inhibiting the CSF-1 receptor on tumor-infiltrating monocytes may enhance the efficacy of radiotherapy for glioblastoma multiforme
Authors: Stafford Jason H. , Chernikova Sophia , West Brian L. , Brown J. Martin
Published: May 2015

Glioblastoma (GBM) is the most common brain tumor and is one of the most deadly forms of cancer with median survival only 15 months after diagnosis. Ionizing radiation (IR) is the most effective treatment for GBM, but the prognosis remains poor because the tumors invariably recur. We have previously shown that bone marrow-derived monocytes promote GBM recurrence after radiotherapy by homing to the irradiation site and stimulating the growth of new tumor blood vessels in a process referred to as vasculogenesis. We found that this infiltration of monocytes can be prevented and tumor recurrences delayed by inhibition of the SDF-1/CXCR4 chemokine axis. Here we present data that suggest Colony Stimulating Factor-1 (CSF-1) may also play an important role in this phenomenon. CSF-1 is a potent chemokine secreted by several different types of solid tumors and is also the main growth factor regulating the differentiation of monocytes into macrophages. RT-PCR analysis of U251 GBM tumors growing in mice revealed a more than 12.5-fold increase in expression of CSF-1 after treatment with a single dose of 12 Gy IR. Increased CSF-1 expression in these tumors correlated with increased recruitment of CD11b+ monocytes. Immunohistochemistry (IHC) and fluorescence activated cell sorting (FACS) showed that the number of CD11b+ monocytes in GBM tumors had approximately doubled within 2 weeks of IR treatment. F4/80 staining indicated that the majority of the CD11b+ cells were mature macrophages. Treatment with 40 mg/kg/day PLX3397, which inhibits the receptor for CSF-1 (CSF-1R), effectively blocked the recruitment of monocytes/macrophages to the irradiated tumors and inhibited tumor blood vessel formation. Moreover, bioluminescence imaging (BLI) of intracranial U251-luciferase tumors growing in mice showed that combined treatment with IR and PLX3397 was more effective at inhibiting tumor growth than IR alone. Eighty percent of mice treated with IR and PLX3397 were alive at 50 days post-irradiation whereas there were no survivors among groups receiving monotherapy. These results suggest inhibition of CSF-1R may prevent post-irradiation recurrence of GBM by blocking the recruitment of tumor-infiltrating monocytes/macrophages.

Citation Format: Jason H. Stafford, Sophia Chernikova, Brian L. West, J. Martin Brown. Inhibiting the CSF-1 receptor on tumor-infiltrating monocytes may enhance the efficacy of radiotherapy for glioblastoma multiforme. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B71. doi:10.1158/1538-7445.CHTME14-B71
Abstract IA04: Cancer-promoting immunity
Authors: Pittet Mikael
Published: May 2015

Tumors often promote the expansion and recruitment of a variety of circulating immune cells. Among them, macrophages can accumulate in large numbers in the tumor stroma and participate actively in cancer growth; however, our understanding of the origins and dynamics of these cells remains not fully explored. By using various modalities, including in vivo imaging, we have started to define where, when and how macrophage precursors are produced and traffic to the tumor stroma. The findings suggest new cancer-induced mechanisms that foster tumor-promoting immunity and identify novel cellular and molecular targets for therapeutic intervention.

Citation Format: Mikael Pittet. Cancer-promoting immunity. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA04. doi:10.1158/1538-7445.CHTME14-IA04
Abstract IA15: Overcoming tumor resistance to VEGF blockade by co-targeting ANG2/TIE2 signaling and macrophages
Authors: Palma Michele De
Published: May 2015

Angiopoietin-2 (ANG2/ANGPT2) is a proangiogenic factor that promotes endothelial-cell survival and growth by activating the TIE2 receptor. We and others have shown that ANG2 blockade inhibits tumor angiogenesis and progression in mouse models of cancer. However, it is unclear whether ANG2 may limit tumor sensitivity to vascular-endothelial growth factor (VEGF)-targeted therapies. By employing mouse tumor models that are either sensitive or insensitive to VEGF inhibition, we found that the efficacy of VEGF signalling blockade may be limited by the adaptive upregulation of ANG2 in the resistant tumors. Indeed, combined ANG2/VEGF signaling blockade suppressed tumor angiogenesis and progression in anti-VEGF-resistant tumors, without eliciting increased tumor invasion or metastasis. These findings clearly strengthen the clinical rationale for co-targeting ANG2 and VEGF in tumors that develop resistance to VEGF inhibition. Notably, a minority of the tumors revascularized after VEGF/ANG2 blockade, showing adaptive resistance to the double antiangiogenic treatment. Because we noted an increase of tumor-associated macrophages (TAMs) in association with resistance to dual VEGF/ANG2 blockade, we have explored the opportunity to combine effective antiangiogenic therapy with pharmacological TAM depletion. The results of these studies will be presented at the meeting.

Citation Format: Michele De Palma. Overcoming tumor resistance to VEGF blockade by co-targeting ANG2/TIE2 signaling and macrophages. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA15. doi:10.1158/1538-7445.CHTME14-IA15
Abstract A19: Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines
Authors: Noesel Max M. van , Duijkers Floor A M , Menezes Renee X. de , Stumpel Dominique J. P. M. , Admiraal Pieter , Pieters Rob , Meijerink Jules P P
Published: May 2015

Background: Epigenetic alterations are inherent to cancer cells, and epigenetic drugs are currently primarily used to treat hematological malignancies. Pediatric neuro-ectodermal tumors originate from neural crest cells and also exhibit epigenetic alterations involving e.g. apoptotic pathways, which suggests that also these tumors may be sensitive to epigenetic drugs. This notion prompted us to assess molecular and functional effects of low dosage epigenetic drugs in neuro-ectodermal tumor-derived cell lines of pediatric origin.

Results: In 17 neuroblastoma (NBL) and 5 peripheral primitive neuro-ectodermal tumor (PNET) cell lines a combination treatment of 5-aza-2′-deoxycytidine (DAC) and Trichostatin A (TSA) at nanomolar dosages was found to reduce proliferation and to induce wide-spread DNA demethylation, accompanied by major changes in gene expression profiles. Approximately half of the genes that were significantly up-regulated upon treatment exhibited a significant demethylation in their promoter regions. In the NBL cell lines, almost every cellular pathway (193/200) investigated showed expression alterations after treatment, especially a marked up-regulation of genes in the p53 pathway. The combination treatment also resulted in up-regulation of known epigenetically regulated genes such as X-chromosomal genes, tissue-specific genes and a limited number of imprinted genes, as well as known tumor suppressor genes and oncogenes.

Conclusions: Nanomolar dosages of epigenetic drugs have a dramatic impact on the genomes of neuro-ectodermal tumor derived cell lines, including alterations in DNA methylation and concomitant alterations in gene expression.

Citation Format: Max M. van Noesel, Floor A M Duijkers, Renee X. de Menezes, Dominique J. P. M. Stumpel, Pieter Admiraal, Rob Pieters, Jules P P Meijerink. Epigenetic drug combination induces genome-wide demethylation and altered gene expression in neuro-ectodermal tumor-derived cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A19.
Abstract A33: Exploring pediatric cancer genomics with the UCSC Cancer Genomics Browser
Authors: Cline Melissa , Morozova Olena , Swatloski Teresa , Craft Brian , Goldman Mary , Haussler David , Zhu Jingchun
Published: May 2015

The UCSC Cancer Genomics Browser (https://genome-cancer.ucsc.edu) is a set of web-based tools to display, investigate and analyze cancer genomics data and associated clinical data. Experimental quantities such as gene expression levels, copy number variation and somatic mutations are displayed next to clinical features such as age of onset and cancer subtype. Users can interactively group or sort data by clinical features to dynamically explore how genomic aberrations relate to clinical outcomes. Integrated Kaplan–Meier plots help investigators assess how clinical or genomic values impact long-term survival. The browser currently hosts data from 144 cancer studies including TCGA, CCLE and LINCS. It can display data from multiple studies at once, facilitating cross-cancer comparisons.

We are currently interested in hosting new pediatric cancer datasets, and can offer either public or protected access as appropriate.

Figure 1 illustrates the power of this approach. It compares patterns of somatic mutations in pediatric high-risk neuroblastoma, a cancer of the peripheral nervous system (Pugh, Morozova et al, Nature Genetics 2013) to those in two adult cancers of the central nervous system: lower grade glioma (LGG, TCGA) and glioblastoma multiforme (GBM, TCGA), and contrasts the age of the patient with frequency of mutations in the Alpha Thalassemia/Mental Retardation Syndrome X-linked (ATRX) gene. The data for all three cancers is sorted by the age of the patient, with yellow being the oldest. The age ranges overlap: the oldest neuroblastoma patients are 16, while the youngest LGG and GBM patients are 14 and 21 respectively. While the three cancers show distinct patterns of somatic mutations, all three show frequent mutations in the ATRX gene in older teens or young adults.

This suggests that these cancers may share age-related subtypes with similar genomic signatures. These subtypes may indicate the age-dependent importance of ATRX in the development of both central and peripheral nervous systems, and may ultimately highlight common therapeutic avenues for the two groups of diseases.

This data set can be explored at the UCSC Cancer Genomics Browser at https://genome-cancer.ucsc.edu/proj/site/hgHeatmap/#?bookmark=pc

Citation Format: Melissa Cline, Olena Morozova, Teresa Swatloski, Brian Craft, Mary Goldman, David Haussler, Jingchun Zhu. Exploring pediatric cancer genomics with the UCSC Cancer Genomics Browser. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A33.
Abstract A45: Autocrine and paracrine IL-6 and IL-8 drive osteosarcoma metastasis
Authors: Roberts Ryan D. , Bid Hemant K. , Phelps Doris , Hendrickson Brett W. , Cam Hakan , Houghton Peter J.
Published: May 2015

For patients with most types of solid tumors, metastases drive the morbidity and mortality of their disease. For patients with osteosarcoma this is particularly true, with metastases that occur almost exclusively within the lung. We set out to identify the factors that facilitate growth of osteosarcoma specifically within the lung environment. To do this, we identified a set of candidate genes and tested them for reliably enriched levels of expression in metastatic lesions relative to primary tumors in the same patients. IL-6 and IL-8 are among the most reliably enriched genes in the metastatic lesions. Osteosarcoma cell lines that express high-levels of IL-6 and IL-8 not only respond to these signals in an autocrine manner, but form lung metastases much more readily than cell lines with low IL-6 and IL-8 expression in mouse xenograft models. IL-6 and IL-8 drive chemokinesis, directional migration, and invasive activity in osteosarcoma cells independent of the degree to which they express these cytokines. Blocking IL-6 and IL-8 signals reduces the metastatic efficiency of osteosarcoma cells. These data suggest that IL-6 and IL-8 play a central role in the mechanisms that account for lung tropism in osteosarcoma and provide attractive targets for therapies intended to prevent metastasis in order to improve outcomes in patients with osteosarcoma.

Citation Format: Ryan D. Roberts, Hemant K. Bid, Doris Phelps, Brett W. Hendrickson, Hakan Cam, Peter J. Houghton. Autocrine and paracrine IL-6 and IL-8 drive osteosarcoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A45.