bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A64: Cell-extrinsic effects of metformin in the breast cancer microenvironment
Authors: Williams Christopher Charles , Miller Kabrina
Published: May 2015

Currently, few drugs have been formulated to disturb the autocrine/paracrine communication circuits which maintain the tumor-supportive microenvironment as a method of cancer chemoprevention. PGE2 (Prostaglandin E2) plays an important role in mediating the production of pro-inflammatory cytokines associated with tumor progression and metastasis. Recently, the anti-diabetic drug metformin has been associated with decreased incidence of several solid cancers among diabetic patients and has previously been shown to reduce expression of systemic inflammatory markers. However, few studies have detailed the intersection of metformin-mediated chemoprevention and chronic inflammation. As such, we investigated whether metformin impacts the inflammatory microenvironment in breast cancer to disrupt tumorigenesis and cancer progression. We hypothesize that metformin disrupts pro-neoplastic autocrine/paracrine inflammatory signaling by BCCs and its associated stroma. We have previously published that metformin inhibits proliferation of breast cancer cells, and that this impairment was associated with increased expression of growth differentiation factor 15 (GDF15) and repression of cyclooxygenase 1 mRNA expression. Here, we confirm that metformin decreases the intracellular and secreted GDF15 levels, and causes a reduction in the expression of proinflammatory annexin A2 protein, a component of the plasminogen receptor. Furthermore, exposure of breast cancer cells to metformin resulted in decreased PGE2 synthesis. Significantly, metformin mediated cell death was abrogated by addition of exogenous PGE2, suggesting that metformin at least in part elicits its antineoplastic function through PGE2 dependent mechanisms. Together these findings suggest a model in which metformin represses annexin A2 and cyclooxygenase expression, culminating in the loss of tumor-associated PGE2, induction of GDF15, and impaired breast cancer cell survival and invasiveness. As such, metformin may function to condition the pre-cancerous microenvironment/stroma to render it inhospitable toward further cancer progression.

Citation Format: Christopher Charles Williams, Kabrina Miller. Cell-extrinsic effects of metformin in the breast cancer microenvironment. [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 A64. doi:10.1158/1538-7445.CHTME14-A64
Abstract A67: Stromal expression of microRNA-21 identifies high-risk group in triple negative breast cancer
Authors: MacKenzie Todd , Schwartz Gary , Wells Wendy , Sempere Lorenzo
Published: May 2015

Introduction: Triple negative breast cancer (TNBC) is an aggressive subtype defined by lack of ER, PR and HER2 expression. There are few specific prognostic markers and no effective targeted therapies for TNBC. microRNA diagnostics is an emerging area of novel biomarkers in cancer medicine. Expression of tumor promoting miR-21 is frequently altered in BC. Since tumor lesions are a complex mixture of different cell types, we hypothesized that analysis of miR-21 expression at single cell resolution could provide more accurate information to assess risk of disease recurrence and BC-related death.

Experimental procedure: We implemented a fully-automated tissue slide-based staining assay in a CLIA-certified environment to detect miR-21 expression in a cohort of 988 BC patients, whose tumors were represented in the NCI CDP Stage I/II Prognostic tissue microarrays. 901 cases provided adequate tissue to determine levels and cellular compartment (cancer vs. stroma) of miR-21 expression.

Results: The miR-21High group had a shorter recurrence-free (RFS; HR=1.71, p<0.001) and BC-specific (CSS; HR=1.96, p<0.001) survival in multivariate regression analyses, adjusted for standard clinicopathologic characteristics. Subdivision of the miR-21High group by cellular compartment of expression revealed that cancer cell-specific expression in ER+ and/or PR+ HER2- cases (HR=3.67, p=0.006 for RFS; HR=5.13, p=0.002 for CSS) and stromal cell expression in ER-PR-HER2- cases ( HR=2.59, p=0.013 for RFS; HR=3.37, p=0.003 for CSS) were associated with very poor clinical outcome.

Conclusions: These findings indicate that levels and cellular compartment of miR-21 expression provide clinically-relevant information in different intrinsic molecular BC subtypes. Importantly, miR-21 expression is predominantly upregulated in the stroma and not cancer cells. This suggests a regulatory role of miR-21 in cellular elements of the tumor microenvironment, including tumor-associated fibroblasts. miR-21 could become a useful functional biomarker to further refine individual risk and offer a new therapeutic avenue to improve management of TNBC.

Citation Format: Todd MacKenzie, Gary Schwartz, Wendy Wells, Lorenzo Sempere. Stromal expression of microRNA-21 identifies high-risk group in triple negative breast 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 A67. doi:10.1158/1538-7445.CHTME14-A67
Abstract A68: GPCRs as potential therapeutic targets in pancreatic cancer-associated fibroblasts
Authors: Zhou Shu , French Randall , Lowy Andrew M. , Insel Paul A.
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stromal matrix, composed of activated fibroblasts/stellate cells, inflammatory cells and other cell types. This unique tumor microenvironment has been increasingly recognized as a key mediator of PDAC progression and drug resistance. Thus, targeting the tumor stroma may be a novel therapeutic approach for PDAC. We hypothesized that G protein-coupled receptors (GPCRs) expressed by pancreatic cancer-associated fibroblasts (CAFs) may be potential therapeutic targets for PDAC. To begin to test this hypothesis, we used an unbiased GPCRomic array approach to identify and quantify the GPCRs expressed by pancreatic CAFs. We found 125 GPCRs, whose expression was shared in CAFs obtained from the primary tumors of five patients. Among those GPCRs, 35 had at least two-fold higher expression in the CAFs compared to normal pancreatic fibroblasts and included GPCRs that link to each of the major classes of G proteins. RT-PCR analysis of the 10 most up-regulated GPCRs correlated well (r2=0.88) with the data from the GPCR array. We conclude that: 1) GPCRomic array analysis can identify and quantify the profile of GPCRs expressed by pancreatic CAFs; 2) the CAF-expressed profile differs from that of normal pancreatic fibroblasts and 3) GPCRs expressed by CAFs may have functional roles and be novel therapeutic targets for PDAC.

Citation Format: Shu Zhou, Randall French, Andrew M. Lowy, Paul A. Insel. GPCRs as potential therapeutic targets in pancreatic cancer-associated fibroblasts. [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 A68. doi:10.1158/1538-7445.CHTME14-A68
Abstract B03: Hypoxia-induced carbonic anhydrase IX promotes MDSC recruitment and establishment of the breast cancer premetastatic niche by stimulating G-CSF production
Authors: Chafe Shawn C. , Lou Yuanmei , Sceneay Jaclyn , Vallejo Marylou , McDonald Paul C. , Möller Andreas , Dedhar Shoukat
Published: May 2015

The recruitment of bone marrow-derived cells (BMDCs) to distant tissue sites prior to the arrival of disseminated tumor cells has been shown to facilitate metastasis in a preclinical setting through the establishment of the pre-metastatic niche. Primary tumor hypoxia has been shown to play a pivotal role in the production of chemokines and cytokines responsible for the mobilization of these BMDCs, especially in breast cancer. Carbonic anhydrase IX (CAIX) expression is highly upregulated by breast cancer cells in hypoxia where its activity contributes to cell survival through the buffering of intracellular pH. Preclinically, CAIX has been demonstrated to be required for breast tumor growth and metastasis, whereas clinically, CAIX is an established indicator of poor prognosis and distant metastasis in breast cancer patients. However, the mechanism by which CAIX exerts its pro-metastatic function is incompletely understood. We investigated whether CAIX function in the hypoxic niche of the primary tumor was involved in establishment of the pre-metastatic niche through the production of soluble mediators of this process. Using shRNA-mediated depletion of CAIX in the metastatic 4T1 murine breast cancer cells and orthotopic tumors, we demonstrate that CAIX is required for the mobilization and recruitment of myeloid-derived suppressor cells (MDSCs) to pre-metastatic niches through the stimulation of granulocyte colony stimulating factor (G-CSF) production, a potent stimulator of MDSC mobilization and recruitment to pre-metastatic niches. We further demonstrate that CAIX expression is required for the activation of NF-κB by hypoxic breast cancer cells and that constitutive activation of the NF-κB pathway upon reducing CAIX expression by shRNA restores G-CSF secretion by hypoxic breast cancer cells. This is the first demonstration of a role for CAIX in the pre-metastatic phase of breast cancer metastasis and indicates that CAIX regulates the expression of G-CSF and recruitment of granulocytic MDSCs to pre-metastatic niches.

Citation Format: Shawn C. Chafe, Yuanmei Lou, Jaclyn Sceneay, Marylou Vallejo, Paul C. McDonald, Andreas Möller, Shoukat Dedhar. Hypoxia-induced carbonic anhydrase IX promotes MDSC recruitment and establishment of the breast cancer premetastatic niche by stimulating G-CSF production. [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 B03. doi:10.1158/1538-7445.CHTME14-B03
Abstract B12: Molecular mechanisms of HIF-1a and HIF-2a activation in nonmalignant cells at the premetastatic niche: Effects on metastatic success
Authors: Branco-Price Cristina , Palazon Asis , Xie Jessica , Johnson Randall S.
Published: May 2015

Tumor hypoxia and the hypoxia-inducible transcription factors (HIF) have been thoroughly characterized in solid tumors, and they are active participants in pathways promoting tumor growth and dispersion.

However, despite their well-known pro-tumorigenic role when stabilized in cancer cells, HIF activation in other cell types has been shown to be less consistent and often antagonistic. We have previously shown that specific deletion of HIF-1a in endothelial cells results in reduced tumor cell migration and metastatic incidence, whereas deletion of HIF-2a in this same cell type has the opposite effect. This phenomenon also involved endothelial-specific nitric oxide production via the inducible NOS (iNOS).

Endothelial cells are at the interface between the primary tumor and the rest of the body, and play a role not only in tumor maintenance and growth, but also in metastatic success and proliferation at secondary sites.

Immune/inflammatory cells are also known to be active players in tumor cell motility and establishment. We are interested in understanding the role of non-malignant cell types in the metastatic process, specifically

(i) signals that trigger HIF-1a or HIF-2a activation at metastatic sites and (ii) how does the stabilization of either HIF isoform (or both) affect tumor cell recruitment and establishment.

This work uses primary cells isolated from different genotypic backgrounds to dissect the hypoxia-response regulatory pathways, using a combination of screenings: transcription factor activation and downstream transcriptional targets, in order to build a map of HIF-dependent metabolism. Specifically, we are interested in the pathways which activation or silencing could have an effect on vascular morphology, permeability, inter-cellular signaling and post-extravasation events. Complementary studies in physiological models of extravasation were used for histological, molecular and biochemical analysis of hypoxic tissue to study molecular players identified by our initial ex-vivo screening.

Citation Format: Cristina Branco-Price, Asis Palazon, Jessica Xie, Randall S. Johnson. Molecular mechanisms of HIF-1a and HIF-2a activation in nonmalignant cells at the premetastatic niche: Effects on metastatic success. [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 B12. doi:10.1158/1538-7445.CHTME14-B12
Abstract B28: Autotaxin, LPA1 and LPA5 receptors in the tumor microenvironment determine melanoma invasion and metastasis
Authors: Lee Sue-Chin , Fujiwara Yuko , Liu Jianxiong , Yue Junming , Tsukahara Ryoko , Szabo Erzsebet , Patil Renukadevi , Miller Duane D. , Balazs Louisa , Oravecz Tamas , Tigyi Gabor J.
Published: May 2015

Autotaxin (ATX), a lysophospholipase D that generates the growth factor-like mediator lysophosphatidic acid LPA, is up-regulated in metastatic and chemotherapy-resistant carcinomas. Many cancer cells secrete ATX, and it contributes to their invasive properties. The biological functions of ATX are mediated by LPA acting on G protein-coupled receptors (LPAR1-8). Many cancers overexpress multiple subtypes of LPAR and the overexpression of LPAR leads to malignant transformation, metastasis, and resistance to therapy. We have previously shown that knockdown of ATX expression in B16F10 melanoma cells decreased tumor invasion in vitro and lung metastasis in vivo. In our present study, we sought to understand the roles of LPAR both in the tumor cells and stromal cells of the tumor microenvironment.

B16F10 melanoma cells predominantly express the LPA5 receptor. We found that these cells do not invade across a matrigel layer in response to LPA. shRNA knockdown of LPA5 receptors in B16F10 attenuates this inhibition, suggesting that the LPA5 receptor acts as an anti-invasive receptor in these tumor cells. Interestingly, we identified a different role for host LPA5 receptor. We found that the incidence of B16F10 lung metastasis was significantly reduced in the LPA5 knockout mice compared to wild type mice. In addition, LPA1 knockout mice also showed diminished B16F10 lung metastasis suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of these knockout animals was apparent at 24 hours after injection. However, deletion of LPA1 or LPA5 did not affect the subcutaneous growth of B16F10 tumors.

In summary, our results suggest that LPAR, in particular LPA5 and LPA1 receptors in tumor and stromal cells may play different roles in invasion and the seeding of metastasis.

Citation Format: Sue-Chin Lee, Yuko Fujiwara, Jianxiong Liu, Junming Yue, Ryoko Tsukahara, Erzsebet Szabo, Renukadevi Patil, Duane D. Miller, Louisa Balazs, Tamas Oravecz, Gabor J. Tigyi. Autotaxin, LPA1 and LPA5 receptors in the tumor microenvironment determine melanoma invasion and metastasis. [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 B28. doi:10.1158/1538-7445.CHTME14-B28
Abstract B33: Langerhans cells prevent melanocytic nevus development and transformation in mice.
Authors: Nasti Tahseen H. , Tsuruta Yuko , McKay Kris , Athar Mohammad , Elmets Craig , Timares Laura
Published: May 2015

The function of Langerhans cells (LCs) in the skin immune system is unclear, and a matter of intense debate. In different mouse models LCs have been shown to induce antitumor immunity or promote skin squamous cell carcinoma (SCC). Their role in the control or promotion of melanoma is not known. We assessed LC's role in the development of DMBA-TPA induced pigmented benign and transformed lesions in a novel mouse model. Transgenic HuLang-DTA+/- C57Bl/6 mice, which selectively ablate epidermal LCs, were crossed with C3H/HeN mice to generate F1 LC knockout (KO) and WT littermate controls. LC-KO mice developed 40% more and 2-fold larger melanocytic lesions than WT. Cell suspensions from LC-KO pigmented skin lesions contained increased CD4+ Foxp3+ regulatory T cells and CD11b-/loGr-1hi cells, consistent with an immunosuppressive microenvironment. CD11c+CD11b+Ly6C+ APCs or MHCII+ cell numbers were fewer in lesions from LC-KO as compared to WT mice. Nests of melanocytic cells were observed at a higher frequency in draining lymph nodes (dLNs) from LC-KO as compared to WT; DLN cells contained increased numbers of TRP2+ cells and routinely gave rise to proliferative melanocytic cell lines. Real-time PCR analysis of epidermal cDNA from TPA treated LC-KO, as compared to WT, demonstrated a profound loss of IL-10, IL-12p35 and IL-12p40, indicating LCs as the major source. Further, IL-23 steady state levels in the epidermis of LC-KO are increased greater than 3-fold over WT, indicating that LCs normally suppress keratinocyte production of IL-23. Thus, in contrast to SCC, LCs inhibit development of benign and metastatic pigmented tumors. This novel murine model will be useful in elucidating transformation mechanisms and identifying novel therapeutic targets that may prevent metastatic melanoma.

Citation Format: Tahseen H. Nasti, Yuko Tsuruta, Kris McKay, Mohammad Athar, Craig Elmets, Laura Timares. Langerhans cells prevent melanocytic nevus development and transformation in mice.. [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 B33. doi:10.1158/1538-7445.CHTME14-B33
Abstract B43: Intratumoral heterogeneity of ovarian cancer stem cells and their interactions with the tumor microenvironment
Authors: Tzukerman Maty , Abelson Sagi , Shamai Yeela , Berger Liron , Skorecki Karl
Published: May 2015

Intratumoral heterogeneity challenges existing paradigms for anti-cancer therapy. We have previously demonstrated that the human embryonic stem cells (hESC)-derived cellular microenvironment in immunocompromised mice, enables functional distinction of heterogeneous tumor cells, including cells which do not grow into a tumor in conventional direct tumor xenograft platform. We have identified and characterized six cancer cell subpopulations each clonally expanded from a single cell, derived from human ovarian clear cell carcinoma of a single tumor, to demonstrate striking intratumoral phenotypic heterogeneity that is dynamically dependent on the tumor growth microenvironment. These cancer cell subpopulations, characterized as cancer stem cell subpopulations, faithfully recapitulate the full spectrum of histological phenotypic heterogeneity known for human ovarian clear cell carcinoma. Each of the six subpopulations displays a different level of morphologic and tumorigenic differentiation wherein growth in the hESC-derived microenvironment favors growth of CD44+/aldehyde dehydrogenase positive pockets of self-renewing cells that sustain tumor growth through a process of tumorigenic differentiation into CD44-/aldehyde dehydrogenase negative derivatives. Strikingly, these derivative cells display microenvironment-dependent plasticity with the capacity to restore self-renewal markers and CD44 expression. We delineated the distinct gene expression and epigenetic profiles of two such subpopulations, representing extremes of phenotypic heterogeneity in terms of niche-dependent self-renewal and tumorigenic differentiation. By combining Gene Set Enrichment, Gene Ontology and Pathway-focused array analyses with methylation status and secretome analyses, we propose a suite of robust differences in tumor self-renewal and differentiation pathways, and interaction with stromal cells in the tumor microenvironment that underlay the striking intratumoral phenotypic heterogeneity which characterized this and other solid tumor malignancies.

Citation Format: Maty Tzukerman, Sagi Abelson, Yeela Shamai, Liron Berger, Karl Skorecki. Intratumoral heterogeneity of ovarian cancer stem cells and their interactions with the tumor microenvironment. [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 B43. doi:10.1158/1538-7445.CHTME14-B43
Abstract B44: The microRNA-200/Zeb1 axis regulates ECM-dependent Src signaling, cytoskeletal changes and cancer cell invasion
Authors: Ungewiss Christin , Roybal Jonathon D. , Peng David H. , Gibbons Don L.
Published: May 2015

The microRNA-200 family (miR-200) has been shown to be a master regulator of epithelial-to-mesenchymal transition (EMT), partially through a double-negative feedback loop with the transcription repressor Zeb1, allowing cells to acquire an invasive and metastatic phenotype. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we are investigating the role of the Zeb1/miR-200 axis in regulating cytoskeletal reorganization, a characteristic requirement of cell motility and invasion. Functional assays demonstrated differential actin reorganization within cell lines with different miR-200 levels based upon the extracellular matrix composition, leading us to hypothesize that miR-200 regulates the activation of signaling pathways that mediate a pro-invasive cytoskeletal reorganization. Based upon the importance of cell-matrix interactions in producing cellular activation we identified the Src pathway as a critical mediator of invasion upon miR-200 repression in murine and human lung cancer cell lines. Pharmacologic inhibition of Src significantly reduces in vitro cell migration and invasion in Boyden chamber assays and in 3D cell cultures, which can be mimicked using Integrin β1 blockade. Biochemical analysis of this Src inhibition reveals a significant reduction in the phosphorylated FAK and cortactin levels, two downstream targets of Src. TGFβ stimulation is a strong EMT inducer, producing miR-200 repression and Src activation in our models, which can be blocked biochemically using Src inhibitors, with associated phenotypic changes in 2D and 3D cultures. This data suggests that miR-200 regulates the cell-matrix contact (e.g. integrins), acting through the Src pathway and modulating central players in actin dynamics to promote actin reorganization and invasion.

Citation Format: Christin Ungewiss, Jonathon D. Roybal, David H. Peng, Don L. Gibbons. The microRNA-200/Zeb1 axis regulates ECM-dependent Src signaling, cytoskeletal changes and 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 B44. doi:10.1158/1538-7445.CHTME14-B44
Abstract B57: Quantifying the contribution of matrix metalloproteinases to the development of the pre-metastatic niche in the lung and liver microenvironments
Authors: Hallett Miranda A. , Dozier E. Ashley , Roland Joseph T. , Fingleton Barbara
Published: May 2015

The leading cause of cancer mortality is metastasis of a primary tumor to distant organs. It is thought that stromal micro-environmental changes occur at these secondary sites before the arrival of tumor cells, which facilitate future colonization and outgrowth. This occurrence is commonly termed as the formation of the “pre-metastatic niche.” Specific factors altered at pre-metastatic niches have already been described in literature (Nature. 2005 Dec 8;438(7069):820-7). These include VEGFR-1, VLA-4 and Fibronectin. Matrix Metalloproteinases (MMPs), known for their involvement in tumor invasion and metastasis, have also been implicated in niche formation. Our goals are to (1) determine the roles of MMPs in generation of the pre-metastatic niche and (2) develop a method of quantification for these niches in the lung utilizing a malignant breast cancer type.

In a distant murine LLC-implantation model, we developed immunofluorescent (IF) staining techniques to define pre-metastatic niches within the lung based on the molecules identified by Kaplan et al. Within this same model, qRT-PCR indicated a substantial increase in lung MMP9 (Gelatinase B) gene expression. This led us to reproduce the pre-metastatic niche within a breast cancer model using a time-coursed malignant murine 4T1-GFP implantation model within Balb/c mammary glands. Visualization was again achieved by IF staining of known factors obtained from Kaplan et al. We utilized these models and the clusters formed to create an algorithm for quantifying the pre-metastatic niche. To confirm this algorithm is specific for premetastatic niches in metastatic tumor models, we also implanted 4T1-GFP cells into MMP9-/- Balb/c mice. These mice result in unorganized protein expression and lack of cluster formation. In addition we utilized a non-metastatic mammary tumor model, 67NR also implanted in Balb/c mice to serve as a negative control for our algorithm. Thus, to confirm the pre-metastatic niche is specific to metastatic tumor models.

Eventually, pre-metastatic MMP activity may indicate potential roles and markers for metastatic spread. Further, this algorithm will provide a new quantitative standard for the field. Thus, this algorithm has translational potential as a test for specific drug efficacy.

Citation Format: Miranda A. Hallett, E. Ashley Dozier, Joseph T. Roland, Barbara Fingleton. Quantifying the contribution of matrix metalloproteinases to the development of the pre-metastatic niche in the lung and liver microenvironments. [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 B57. doi:10.1158/1538-7445.CHTME14-B57
Abstract B65: Proteolytic shedding of the cell adhesion molecule ALCAM generates a spatial and context-dependent heterogeneity important for metastasis.
Authors: Hansen Amanda G. , Hebron Katie , Palmer Trenis D. , Arnold Shanna , Zijlstra Andries
Published: May 2015

Heterogeneity in the tumor microenvironment is dictated in part by context-dependent changes in cell-cell interactions. Subpopulations of tumor cells can responding to local micro-environmental changes in a manner that distinguishes them biochemically as well as behaviorally from the rest of the tumor. We identified such a phenomenon in the proteolytic shedding of the cell adhesion molecule ALCAM (Activated Leukocyte Cell Adhesion Molecule). ALCAM controls cell adhesion in a variety of cell types including T-cells, epithelial cells, neurons and endothelial cell. Although ALCAM mRNA is detected in all epithelial tissues and the tumors they give rise to, the detection of cell-surface ALCAM is highly variable, being strong in some regions of the tumor while completely absent in adjacent tumor cells. Through molecular analysis of cell-surface ALCAM we determined that this adhesion molecule is shed from the surface by ADAM17 in response to cytokines from the local milieu.

Although ALCAM shedding is common in cancer, the loss of ALCAM greatly diminishes metastasis, thereby suggesting that ALCAM remains necessary for tumor progression. Indeed, ALCAM shedding supports a dynamic regulation of cell adhesion which, in turn, supports tumor cell dissemination. Considering that the proteolytic processing of ALCAM is functionally important in tumor progression, the detection of ALCAM shedding should be a molecular indicator of disease progression. Indeed, through analysis of retrospective patient cohorts, we demonstrated ALCAM shedding to be a prognostic indicator of disease progression in cancers of the colon, kidney and bladder.

Our observations demonstrate that spatial and context-dependent heterogeneity can be accomplished through post-translational proteolysis of cell adhesion molecules. This process enables tumor cells to respond dynamically to the local milieu and disseminate to a more hospitable environment. Identifying this proteolysis in cancer patients through the detection of ALCAM shedding can predict metastatic progression and long-term patient outcome.

Citation Format: Amanda G. Hansen, Katie Hebron, Trenis D. Palmer, Shanna Arnold, Andries Zijlstra. Proteolytic shedding of the cell adhesion molecule ALCAM generates a spatial and context-dependent heterogeneity important for metastasis.. [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 B65. doi:10.1158/1538-7445.CHTME14-B65
Abstract B72: Serine/threonine kinase MLK4 is a master regulator for proneural-mesenchymal transformation of glioma stem cells
Authors: Sunghak Kim , Violaine Rosenstiel-Goidts , Cheng Shi-Yuan , Ichiro Nakano
Published: May 2015

High-grade glioma is a highly aggressive form of brain cancer associated with a poor prognosis. Recently, we identified that mesenchymal glioma stem cells (Mes GSCs) of HGG are linked to radiation resistance and proneural (PN) GSCs acquire Mes identity via radiation treatment. Here we performed a combined kinome-wide RNA interference screen with genome wide expression analysis to identify kinase-encoding genes that are essential for mesenchymal identity of GSCs. Through this stepwise screening with patient-derived GSC samples, we identified the serine/threonine kinase MLK4 as a key regulator for Mes GSC survival and cell cycle progression in vitro and tumor initiation and propagation in vivo. In addition, overexpression of MLK4 promotes, while both its gene-specific shRNA and pharmacological inhibition attenuate, proneural-mesenchymal transformation (PMT) of GSCs. In patient-derived GSCs, expression of MLK4 protein positively correlated with endogenous EGFRvIII expression and the in vivo growth of mouse xenografts derived from glioma cells with EGFRvIII expression was prominently inhibited by MLK4 knockdown. MLK4-overexpressing PN GSCs acquired radiation resistance, while MLK4 elimination by shRNA radiosensitized Mes GSCs. Patients with a higher MLK4 expression showed reduced survival in CD44-high populations. Collectively, these data indicate that MLK4 as a plausible target for therapy against Mes GBM as well as post-irradiation PN GBM.

Citation Format: Kim Sunghak, Rosenstiel-Goidts Violaine, Shi-Yuan Cheng, Nakano Ichiro. Serine/threonine kinase MLK4 is a master regulator for proneural-mesenchymal transformation of glioma stem 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 B72. doi:10.1158/1538-7445.CHTME14-B72
Abstract B76: Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma
Authors: Berning Manuel , Boukamp Petra
Published: May 2015

3D in vitro skin models are valuable research tools to investigate normal keratinocyte as well as skin carcinoma cell interaction with dermal fibroblasts. Currently, the models lack either a structured vital human extracellular matrix or long-term stability and longevity. Therefore, we aimed at a new 3D in vitro skin model for normal human epidermis and tumorigenic stages of skin cancer.

This model is based on dermal equivalents containing a highly structured cell-derived matrix (CDM) produced by human dermal fibroblasts. First, we show that the CDM dermal equivalent supports a high structural integrity of the skin model and maintains a vital epidermis for six months. The epidermis is well-stratifying and differentiates in a regular orthokeratotic pattern. Transmission electron microscopy reveals ultrastructural entities as basement membranes, hemidesmosomes, desmosomes, keratohyalin granulae and lamellar bodies. In line, the molecular differentiation markers are properly distributed. Second, five non-tumorigenic and tumorigenic HaCaT keratinocyte variants are tested for their proliferation, differentiation and invasion capacities. After four weeks of co-cultivation the tumorigenic HaCaT variants show hyperproliferative epithelia and lose the capability of an ordered stratification and differentiation. Furthermore, they destroy the basement membrane as well as the extracellular matrix and invade the dermal equivalent. Moreover, seven squamous cell carcinoma (SCC) cell lines were co-cultivated with the CDM dermal equivalent. All SCC cells form hyperproliferative epithelia that heavily invade the extracellular matrix. Remarkably, the epithelia are individually distinct for their invasion and differentiation capacity. Thus, the CDM-based skin cancer model is suitable for demonstrating the heterogeneity of SCC tumors and their interaction with the microenvironment.

In conclusion, the CDM model for normal skin and tumorigenic keratinocytes provides a novel tool to investigate keratinocyte-fibroblast interactions and microenvironment modulations during SCC development. Therefore, this system will provide further insights into mechanistic processes and the interaction of SCCs with their tumor microenvironment.

Citation Format: Manuel Berning, Petra Boukamp. Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma. [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 B76. doi:10.1158/1538-7445.CHTME14-B76
Abstract B85: High malignant prostate cancer cells developed enhanced tumor-initiating/metastatasis-initiating activity and altered the lung microenvironment
Authors: Tzeng Sheue-Fen , Tsai Chin-Hsien , Lee Ming-Shyue , Hsiao Pei-Wen
Published: May 2015

Hormone-refractory and metastatic prostate cancer (PCa) both result in therapeutic failure. Developing PCa animal model is critical to understand the cancer progression and to access therapeutic effects. Here, we established the orthotopic xenograft of androgen-independent PCa cell line, 22Rv1, in nude mouse model and cultured the metastatic cancer cells. The same procedure was repeated to evolve highly metastatic cells, 22Rv1-LN3. In experimental metastasis, 22Rv1-LN3 can colonize and grow in the LN, lung and brain through tail vein injection but 22Rv1 cell failed to colonize in these organs. Besides, intracardiac injection of 22Rv1-LN3 also caused bone metastasis. In the model, we found 22Rv1-LN3 cell have higher protease activity (e.g. matriptase and MMP9) along with lower level of protease inhibitors, (e.g. HAI-2, a matriptase inhibitor and TIMP2/TIMP3). Transcriptional level of these genes determined the in vitro invasion activity and in vivo tumorigenicity. Given the differential capacity to seed different tissues, we further analyze the stem cell property by tumorsphere assay and in vivo transplantation with a reduced cell number. Sphere-formation rate in 22Rv1-LN3 cells was 8% compared to only 0.1% in 22Rv1 cells. Tumorigenicity of 22Rv1-LN3 cells in orthotopic transplantation was 100% (8/8) compared to 20% (2/10) in 22Rv1 cells. Moreover, primary tumor of 22Rv1-LN3 cells developed spontaneous metastases in lymph nodes and lung at 8-week, whereas 22Rv1 cells hardly metastasized even at 14-week. As analyzed by qRT-PCR, primary 22Rv1-LN3 tumor vs. 22Rv1-Luc2 induced higher levels of Arginase and iNOS expression in lung tissue, which indicates the proclivity for metastatic niche formation. In conclusion, this PCa progression model not only elevated the malignant properties of cancer cells but also influenced the tumor microenvironment.

Citation Format: Sheue-Fen Tzeng, Chin-Hsien Tsai, Ming-Shyue Lee, Pei-Wen Hsiao. High malignant prostate cancer cells developed enhanced tumor-initiating/metastatasis-initiating activity and altered the lung microenvironment. [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 B85. doi:10.1158/1538-7445.CHTME14-B85
Abstract IA21: Of niche construction and loss of homeostasis: New insights into the cancer diaspora
Authors: Pienta Kenneth J.
Published: May 2015

Merging the social science concept of diaspora with ecological and population sciences concepts can inform the cancer field to understand the biology of tumorigenesis and metastasis and inspire new ideas for therapy. Together, cancer cells and host cells, interacting within their habitat, create an ecosystem. This ecosystem, in turn, exists within a larger environment (the host patient as biosphere). Cancer cells escape their confinement of their original habitat in the primary tumor, likely forced out by ecological changes in their home niche.

A diaspora is the scattering of people away from an established homeland. To date, “diaspora” has been a uniquely human term utilized by social scientists, however, the application of the diaspora concept to metastasis may yield new biological insights as well as therapeutic paradigms. The diaspora paradigm takes into account and models several variables: the quality of the primary tumor microenvironment, the fitness of individual cancer cell migrants as well as migrant populations, the rate of bidirectional migration of cancer and host cells between cancer sites, and the quality of the target microenvironments to establish metastatic sites.

Once seeded to a new site, diaspora populations do not assimilate into the new environment, they try “recapitulate” their homeland. Niche construction is the process in which an organism alters its own (and consequently, other species) environment, usually in a manner that increases its chances of survival. This creates a feedback relationship between natural selection and niche-construction: when organisms affect their environment, that change can then cause a shift in what traits are being naturally selected for. The effect of niche construction is especially pronounced in situations where environmental alterations persist for several generations, introducing the evolutionary role of ecological inheritance.

Ecological scientific principles can be applied to the cancer diaspora to develop new therapeutic strategies (ecotherapy). It is easier to drain the swamp that the cancer cells created than swat a million cancer cell mosquitos. Alternatively, ecological traps, habitats that lead to the extinction of a species, could be developed to attract cancer cells to a place where they can be better exposed to treatments or to cells of the immune system for improved antigen presentation. Targeting the microenvironment in addition to the cancer cells themselves should improve therapeutic outcomes.

Citation Format: Kenneth J. Pienta. Of niche construction and loss of homeostasis: New insights into the cancer diaspora. [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 IA21. doi:10.1158/1538-7445.CHTME14-IA21
Abstract PR06: Lymphocyte invasion of basal breast tumors is associated with wild-type p53
Authors: Quigley David A. , Vaske Charlie , Silwa-Pandit Laxmi , Dannenfelser Ruth , Langerød Anita , Vollan Hans Kristian M. , Troyanskaya Olga , Chin Suet-Feung , Caldas Carlos , Balmain Allan , Børresen-Dale Anne-Lise , Kristensen Vessela
Published: May 2015

The tumor suppressor p53 is mutated in approximately 30% of breast adenocarcinomas, but the clinical implications of p53 mutation depend on the subtype of breast tumor. Although p53 mutation is associated with worse outcome for patients with Luminal B and Her2 subtypes, the same is not true in Basal tumors, despite the fact that Basal tumors have the highest frequency of p53 mutation of any subtype. Lymphocytic infiltration is associated with better prognosis in several epithelial malignancies including triple-negative breast tumors. In this study we identify an association in Basal breast cancer between lymphocytic infiltration and the number of copies of wild-type p53 by combining gene expression, p53 sequencing, DNA copy number analysis, and semi-quantitative immune cell histology in 1,281 tumors. We use the Nanodissect algorithm to define signatures for T cell types and show that the infiltration consists primarily of cytotoxic T lymphocytes. Elevated expression of this gene signature is associated with better outcome in Basal tumors, and differential pathway activity analysis identifies significant activation of caveolin 1 signaling to TGF-beta in p53 wild-type Basal tumors. These findings identify a new link between the p53 pathway and beneficial adaptive immune response in breast tumors not responsive to the estrogen receptor, supporting a connection between a lack of p53 function and the failure of tumor immunosurveillance.

This abstract is also presented as Poster A35.

Citation Format: David A. Quigley, Charlie Vaske, Laxmi Silwa-Pandit, Ruth Dannenfelser, Anita Langerød, Hans Kristian M. Vollan, Olga Troyanskaya, Suet-Feung Chin, Carlos Caldas, Allan Balmain, Anne-Lise Børresen-Dale, Vessela Kristensen. Lymphocyte invasion of basal breast tumors is associated with wild-type p53. [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 PR06. doi:10.1158/1538-7445.CHTME14-PR06
Abstract PR07: Neutralizing the activity of murine TGF-β receptor 2 promotes a differentiated tumor cell phenotype and inhibits pancreatic cancer metastasis
Authors: Ostapoff Katherine T. , Cenik Bercin Kutluk , Wang Miao , Ye Risheng , Xu Xiaohing , Nugent Desiree , Hagopian Michelle , Topalovski Mary , Rivera Lee B. , Carroll Kyla D. , Brekken Rolf A.
Published: May 2015

Elevated levels of TGF-β are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result the TGF-β pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGF-β remains challenging because TGF-β has tumor suppressor functions in many epithelial malignancies including pancreatic cancer. In fact, direct neutralization of TGF-β promotes tumor progression of genetic murine models of pancreatic cancer. Here we report that neutralizing the activity of murine TGF-β receptor 2 using a monoclonal antibody (2G8) has potent anti-metastatic activity in orthotopic human tumor xenografts, syngenic tumors and a genetic model of pancreatic cancer. 2G8 induced an anti-metastatic stromal phenotype by increasing pro-inflammatory macrophages and NK cells and concomitantly decreasing anti-inflammatory macrophages, MDSCs and activated fibroblasts. These stromal specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGF-β signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGF-β dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors.

This abstract is also presented as Poster A38.

Citation Format: Katherine T. Ostapoff, Bercin Kutluk Cenik, Miao Wang, Risheng Ye, Xiaohing Xu, Desiree Nugent, Michelle Hagopian, Mary Topalovski, Lee B. Rivera, Kyla D. Carroll, Rolf A. Brekken. Neutralizing the activity of murine TGF-β receptor 2 promotes a differentiated tumor cell phenotype and inhibits pancreatic cancer metastasis. [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 PR07. doi:10.1158/1538-7445.CHTME14-PR07
Abstract A32: A specific antibody for Histone H3 Lys27Met mutation for the characterization of pediatric glioblastomas
Authors: Lozsa Anita , Clark Robin T. , Lewis Peter W. , Koletsky Matthew S. , Becher Oren J. , Rosenfeld John , Michael Sturges , Allis David C. , Chuang Trinette , Speckmann Wayne , Solache Alejandra
Published: May 2015

Pediatric gliomas such as glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG) are aggressive brain tumors for which there is no effective therapy. The reason for the poor therapeutic outcome for children diagnosed with glioblastomas might be due to the therapeutic approach, which is not significantly different from the treatment of adult gliomas. Recent studies have shown that the gene expression profile of pediatric gliomas is distinct from histologically similar tumors in adults. These findings strongly suggest that the underlying mechanisms of tumor progression are different in these gliomas. Whole-genome sequencing of these pediatric tumors has revealed mutations in genes of H3F3A and HIST1H3B encoding histone H3.3 or H3.1, respectively. The mutations resulted in a substitution of lysine (K) to methionine (M) at position 27 of the protein (H3K27M). The lysine 27 residue of histone H3 is a posttranslational modification site, methylated by the polycomb repressive complex 2 (PRC2). A mutation like H3K27M might play a significant role in tumorigenesis and tumor progression since histones and their posttranslational modifications are key epigenetic regulators. In collaboration with the laboratory of David Allis, we raised antibodies against the K27M mutation of histone H3. Using this antibody, Lewis et al. corroborated the presence of H3 K27M protein in DIPG samples containing the mutant allele. Furthermore, they provided evidence that this mutation can alter the molecular processes in the nucleus through a gain-of-function mechanism in glioblastoma. Here we show that this Anti-Histone H3, K27M mutant antibody (Cat. No.: ABE419), together with other modified histone antibodies, are useful tools for the characterization and classification of different type of brain tumors and can be used in several applications including immunoblotting and chromatin immunoprecipitation (ChIP).

Citation Format: Anita Lozsa, Robin T. Clark, Peter W. Lewis, Matthew S. Koletsky, Oren J. Becher, John Rosenfeld, Sturges Michael, David C. Allis, Trinette Chuang, Wayne Speckmann, Alejandra Solache. A specific antibody for Histone H3 Lys27Met mutation for the characterization of pediatric glioblastomas. [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 A32.