bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1439: Periostin expression is altered by TGF and hypoxia during breast cancer progression.
Authors: Coleman Michelle , Lance Amanda , Ricci Krista , Secrest Adam , Dréau Didier
Published: August 2014

The extracellular matrix (ECM) contributes to the generation of normal breast tissue, and variations in the composition of the ECM have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Specific ECM proteins are also important in the progression of several human cancers, and in particular upregulation of the ECM protein periostin has been linked to poor outcomes in breast cancer. Here we investigated the expression of periostin in biopsy specimens collected from breast cancer patients, as well as determined the differential effects of TGFß and hypoxia on the expression of periostin mRNA and protein in human breast cancer cells. Immunohistological analyses of the breast cancer biopsy specimens indicate that breast cancer tissues expressed different levels of periostin regardless of the stage of the disease and that the expression of periostin tended to decrease as breast cancer progressed. Multiple splice variants of periostin mRNA were expressed by breast cancer cells as determined by RT-PCR. Furthermore, treatments with either TGFß or hypoxia led to significantly different expression of periostin mRNA isoforms and of total level of periostin protein (p<0.05) by breast tumor cells. These observations support the role of periostin in breast cancer progression and suggest a role for specific periostin isoforms in response to changes in the microenvironment including tissue hypoxia and TGFß paracrine signaling from mesenchymal and immune cells.

[This works is supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007)]

Citation Format: Michelle Coleman, Amanda Lance, Krista Ricci, Adam Secrest, Didier Dréau. Periostin expression is altered by TGF and hypoxia during breast cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1439. doi:10.1158/1538-7445.AM2013-1439
Abstract 145: Finding correlations between CtBP expression and breast cancer risk factors.
Authors: Obadina Mofiyinfoluwa A.
Published: August 2014

In women, breast cancer is ranked second in cancer deaths; almost 40,000 deaths are expected and over 225,000 new cases are expected. Research has shown that breast cancer prevalence and mortality are influenced by various factors including race and lifestyle. Studies have indicated racial disparity whereby young women of African descent have higher breast cancer mortality rate and this could be partly explained by higher occurrence of more aggressive breast cancer forms that exhibit characteristics of epigenetically re-programmed reactivation of embryonic processes that promote tumor invasion, spread and resistance to chemotherapy. These are features common to certain breast cancer subtypes including basal-like and triple negative breast cancers, which occur more frequently in younger African American women. We are focusing on epigenetic factors that explain this disparity as this understanding will help provide population-based approach to detecting and treating breast cancer.

C-terminal Binding Protein (CtBP) is a family of epigenetic regulators that undergoes dimerization which is stimulated and stabilized by NADH, a product of metabolism. CtBP has been shown to induce epithelial-to-mesenchymal transition (EMT), an embryonic program that increases tumor invasion and metastasis; we therefore hypothesize that CtBP likely plays a role in tumor progression. Research from our lab has shown an elevated level of CtBP present in the nuclei of more clinically aggressive breast cancer subtypes..

To find the correlation between CtBP expression and other patient factors such as race, age, BMI and hormone receptor status we have begun to screen several breast cancer patient cohorts obtained from larger minority serving medical centers around the country. Our preliminary findings reveal that elevated levels of CtBP expression in patient tumor samples shows a statistically significant correlation with shortened median survival. Moreover, random sampling of a small population of these cohorts suggests that the correlation between CtBP and poor clinical outcome is greater in women of African heritage compared to European descent. How epithelial features and stromal attributes of the tissue microenvironment contribute to this correlation is currently under investigation.

Citation Format: Mofiyinfoluwa A. Obadina. Finding correlations between CtBP expression and breast cancer risk factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 145. doi:10.1158/1538-7445.AM2013-145
Abstract 1455: In vivo circulating tumor cells in blood, lymph and cerebrospinal fluid: dynamic cross-correlations and amplification for cancer diagnosis.
Authors: Galanzha Ekaterina I.
Published: August 2014

We introduced a new platform of multicolor multi-fluid in vivo flow cytometry for real-time ultrasensitive molecular detection and counting of circulating tumor cells (CTCs) and their subpopulations the blood, lymph and cerebrospinal fluid's (CSF) flows. To achieve these goals we integrated multicolor photoacoustic (PA), photothermal (PT) and fluorescent flow cytometry, and combined this technical platform with multiplex molecular targeting strategy using low-toxicity, gold-based functionalized nanoparticles as PA/PT high-contrast agents or genetically encoded proteins (e.g., green fluorescent protein [GFP]) as fluorescent labels. This platform can provide also label-free detection mode using intrinsic (e.g., melanin) molecular labels. The advantages of this new generation of flow cytometries are (1) improving sensitivity by at least a two-order of magnitude compared to available CTC-assays, and (2) significantly extending biomedical applications compared to existing in vivo flow cytometries.

Using preclinical tumor-bearing mouse models of metastatic melanoma and breast cancer, and spiked human blood samples, we demonstrated that our technology can overcome great challenge in cancer research related to monitoring lymph, CSF and blood CTCs at single-cell levels in the natural environment in vivo over the long-term period of metastatic disease development from an early micrometastatic stage to deadly multiple macro-metastases. The obtained results allowed us to estimate in vivo cross-correlations between lymph, CSF and blood CTC counts, size of primary tumor and metastasis (micrometastasis) progression. We also showed that the presented approach can break some important diagnostic limitations of existing methods (e.g., low sensitivity) and potentially permits prevention, or at least inhibition, of metastatic progression. Taking into account the safe nature of the PA technology, we anticipate its quick translation for use in humans.

Citation Format: Ekaterina I. Galanzha. In vivo circulating tumor cells in blood, lymph and cerebrospinal fluid: dynamic cross-correlations and amplification for cancer diagnosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1455. doi:10.1158/1538-7445.AM2013-1455

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1472: Assessment of CTC-based pharmacodynamic biomarkers in NCI clinical trials of targeted anticancer therapeutics.
Authors: Wang Lihua H. , Kummar Shivaani , Pfister Thomas D. , Balasubramanian Priya , Parchment Ralph E. , Tomaszewski Joseph E. , Doroshow James H. , Kinders Robert J.
Published: August 2014

Circulating tumor cells (CTC) potentially represent a substitute for biopsy in assessing drug effects on targets. We have previously reported development and validation of CTC-based pharmacodynamic (PD) biomarker assays that directly examine drug-induced responses (e.g. a DNA damage marker γH2AX, and a tumor suppressor marker p16/INK4a). Here, we summarize our results to date and assess utility of CTC-based biomarkers in patients undergoing clinical trials of targeted anti-cancer therapeutics. Across eight NCI Phase 1 and 2 trials in a variety of solid tumors, the portion of the population statistically evaluable is approximately 30% for all trials; the CTC biomarker statistical evaluation is limited by the total number of CTCs collected from each tube of blood. From data obtained from multiple trials of topoisomerase1 and PARP inhibitors, the γH2AX-positive CTC baseline level was less than 20% in 34 of 50 patients. The fraction of CTCs expressing γH2AX independent of changes in the total CTC count, increased in patients following treatment with different topoisomerase 1 inhibitors alone or in combination with other drugs. Furthermore, correlations between γH2AX levels and overall responses were demonstrated in patients with refractory cancer in a phase II randomized trial of the combination of Veliparib with metronomic oral cyclophosphamide. Pharmacodynamic studies measuring re-expression of the tumor suppressor p16 in response to the epigenetic therapy regimen of 5-fluoro-2-deoxycytidine plus tetrahydrouridine yielded similar results on the number of patients evaluable using CTCs in the NCI intramural patient population. Given the limitations of surface molecule-based CTC methods, we are developing and evaluating an antibody-independent CTC capture technology to analyze biomarkers, starting in small animals using lower blood volumes and in patients with a variety of advanced epithelial and non-epithelial cancers. Our studies indicate that assessment of CTC-based PD biomarkers has value for rapidly assessing proof of mechanism in the clinical development of molecularly targeted anticancer therapeutics. Funded by NCI contract # HHSN261200800001E

Citation Format: Lihua H. Wang, Shivaani Kummar, Thomas D. Pfister, Priya Balasubramanian, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow, Robert J. Kinders. Assessment of CTC-based pharmacodynamic biomarkers in NCI clinical trials of targeted anticancer therapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2013-1472
Abstract 1475: Mutant p53 is a regulator of NADPH-oxidase 4 (Nox4) in TGF-beta-mediated migration of human lung and breast epithelial cells.
Authors: Boudreau Howard E. , Casterline Benjamin W. , Burke Devin J. , Leto Thomas L.
Published: August 2014

The epithelial-to-mesenchymal transition (EMT) leads to development of increased cell plasticity that occurs normally during wound healing and embryonic development and can be coopted for cancer invasion and metastasis. TGF-beta induces EMT but the mechanism is unclear. We have previously shown that Nox4, a member of the NADPH oxidase (Nox) family, is a source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Recent studies have shown that p53 plays an important role in TGF-beta-mediated signaling and epithelial cell migration. Our current study investigates whether p53 is mechanistically involved in TGF-beta-regulated Nox4 expression and cell migration. Our results indicate that wild-type p53 is a potent suppressor of TGF-beta-induced Nox4 expression, ROS production, and cell migration. In contrast, expression of tumor-associated mutant p53 proteins (R175H or R280K) correlated with enhanced Nox4 expression and cell migration in a TGF-beta dependent manner. Furthermore, knockdown of the endogenous p53 mutant (R280K) in MDA-MB-231 cells by p53-targeted siRNA resulted in decreased Nox4 protein as well as phosphorylated Src and FAK proteins. TGF-beta treatment of MDA-MB-231 cells overexpressing a dominant-negative form of Nox4 also showed a decrease in phosphorylated Src and FAK proteins, thereby indicating mutant p53 (R280K) and Nox4 are regulators of Src and FAK activity. Collectively, our findings suggest important physiological functions for both wild type and mutant p53 proteins in regulating Nox4-dependent signaling in TGF-beta-mediated cell migration.

Citation Format: Howard E. Boudreau, Benjamin W. Casterline, Devin J. Burke, Thomas L. Leto. Mutant p53 is a regulator of NADPH-oxidase 4 (Nox4) in TGF-beta-mediated migration of human lung and breast epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1475. doi:10.1158/1538-7445.AM2013-1475
Abstract 1476: Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β.
Authors: Mele Valentina , Muraro Manuele Giuseppe , Calabrese Diego , Amatruda Nunzia , Amicarella Francesca , Pfaff Dennis , Kvinlaug Brynn , Bocelli-Tyndall Chiara , Martin Ivan , Resink Therese J. , Terracciano Luigi M. , Spagnoli Giulio Cesare , Iezzi Giandomenica
Published: August 2014

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor site. Because of this feature, they are currently being used in preclinical models and in clinical trials as potential vehicles for delivery of targeted anti-cancer therapies. Clinical applications of MSC however, require a deeper understanding of their biology and, in particular, of their interaction with cancer cells. In this study we have investigated the effects mediated by human bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC induced upregulation of epithelial-to-mesenchymal (EMT)-related genes and downregulation of E-cadherin in tumor cells. These effects required cell-to-cell contact and were mediated by expression of surface-bound TGF-β, induced in MSC upon coculture with tumor cells. CRC cells exposed to MSC formed larger tumor masses in vivo, characterized by higher vessel density and increased mesenchymal marker expression, and displayed enhanced invasive capacity. Thus, by promoting cell expansion, EMT-related phenomena and tumor angiogenesis, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells. These findings raise concerns regarding the safety of MSC administration for clinical purposes in patients with cancer.

Citation Format: Valentina Mele, Manuele Giuseppe Muraro, Diego Calabrese, Nunzia Amatruda, Francesca Amicarella, Dennis Pfaff, Brynn Kvinlaug, Chiara Bocelli-Tyndall, Ivan Martin, Therese J. Resink, Luigi M. Terracciano, Giulio Cesare Spagnoli, Giandomenica Iezzi. Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1476. doi:10.1158/1538-7445.AM2013-1476
Abstract 1477: Protein Tyrosine Kinase 6 promotes peripheral adhesion complex formation, cell migration, and the epithelial mesenchymal transition in prostate cancer.
Authors: Tyner Angela L. , Zheng Yu
Published: August 2014

Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is evolutionarily related to Src kinases. In the normal prostate, total and active PTK6 is localized within the nucleus, but nuclear localization is lost in prostate tumors. While the majority of total PTK6 is localized within the cytoplasm of PC3 cells, a human prostate cancer cell line, the active pool of PTK6 that is phosphorylated on tyrosine residue 342 is membrane associated. Ectopic expression of membrane targeted PTK6 leads to enhanced activation of PTK6 at the membrane and formation of peripheral adhesion complexes and increased migration of PC3 cells. Ectopic expression of PTK6 in SYF cells, a nontransformed mouse embryonic fibroblast cell line lacking Src, Yes, and Fyn kinases leads to cell transformation and tumor growth in vivo. Ectopic expression of active membrane-targeted PTK6 in PC3 cells promotes the epithelial mesenchymal transition (EMT). PTK6 regulates the EMT through AKT, as knockdown of AKT or inhibition of AKT activity by knockdown of p130CAS partially rescues the phenotype. PC3 cells expressing active membrane targeted PTK6 exhibit increased migration in vitro and form more metastases in a xenograft mouse model, while knockdown of PTK6 promotes an epithelial phenotype and impairs tumorigenesis in vivo. In prostates of Probasin-Cre, Pten flox/flox mice, striking activation of endogenous mouse PTK6 is observed at the membrane in Pten-null prostates, and this is accompanied by deregulated expression of E-cadherin and vimentin. We found a reverse correlation between PTK6 and E-cadherin expression in normal and metastatic human prostate cancer samples, and show PTK6 is activated at the membrane in some high-grade prostate tumors. Our studies reveal novel functions for PTK6 in prostate cancer, and define it as a potential therapeutic target.

Citation Format: Angela L. Tyner, Yu Zheng. Protein Tyrosine Kinase 6 promotes peripheral adhesion complex formation, cell migration, and the epithelial mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2013-1477
Abstract 1495: Regulation of the epithelial-mesenchymal transition by claudin-3 and claudin-4.
Authors: Shang Xiying , Lin Xinjian , Manorek Gerald , Howell Stephen
Published: August 2014

The mechanisms that control intracellular adhesion are central to the process of invasion and metastasis. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are major structural molecules of the tight junctions that link epithelial cells. Our prior work has demonstrated that knockdown of the expression of either CLDN3 or CLDN4 produces marked changes in the phenotype of ovarian carcinoma cells including increases in growth rate in vivo, migration, invasion, metastasis and drug resistance, similar to those produced by the epithelial-to-mesenchymal transition (EMT). We postulated that these changes may result from the ability of CLDN3 or CLDN4 to suppress EMT. In this study we found that knockdown of either CLDN3 or CLDN4 increased cell size and resulted in elongated, fibroblastic morphology. While knockdown of CLDN3 or CLDN4 did not alter the expression of vimentin, it significantly down-regulated the level of E-cadherin and up-regulated N-cadherin expression. Conversely, over-expression of CLDN3 or CLDN4 in a cell line that does not express endogenous CLDN3 or CLDN4 decreased N-cadherin expression. Re-expression of E-cadherin in the CLDN3 or CLDN4 knockdown cells reduced migration, invasion and tumor growth in vivo. Loss of either CLDN3 or CLDN4 resulted in activation of the PI3K pathway as evidenced by increased Akt phosphorylation, and up-regulation of the mRNA and protein levels of the transcription factor Twist. Taken together, these findings suggest that CLDN3 and CLDN4 function in the ovarian cancer cells to sustain an epithelial phenotype and that their loss promotes EMT.

Citation Format: Xiying Shang, Xinjian Lin, Gerald Manorek, Stephen Howell. Regulation of the epithelial-mesenchymal transition by claudin-3 and claudin-4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1495. doi:10.1158/1538-7445.AM2013-1495

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 15: Association of topoisomerase II expression and cancer-specific death in patients with surgically-resected clear cell renal cell carcinoma.
Authors: Joseph Richard W. , Serie Dan , Hilton Tracy , Parasramka Mansi , Eckel-Passow Jeanette , Cheville John , Leibovich Bradley C. , Parker Alexander S.
Published: August 2014

PURPOSE: Despite the development of prognostic algorithms based on clinico-pathologic features, the ability to identify aggressive forms of clear cell renal cell carcinoma (ccRCC) remains suboptimal. Topoisomerase IIA (TOP2a) is a biomarker of DNA replication and a target for antineoplastic agents. Herein, we evaluate the association of TOP2a expression in ccRCC tumors with pathologic features of aggressiveness and risk of cancer-specific death.

METHODS: We identified 947 patients who underwent nephrectomy to treat clinically localized ccRCC between 1/16/1990 and 9/27/2006. TOP2a expression was assessed using IHC and scored as number of positive cells per mm2. We evaluated TOP2a expression using a continuous variable and tertile categories. For associations with pathologic features we employed Kruskal-Wallis tests and for associations with cancer-specific survival we generated Cox proportional hazard regression models.

RESULTS: Higher TOP2a expression is associated with later stage, higher grade and higher Mayo SSIGN score (all p < 0.001). The risk of death from RCC increases with increasing TOP2a expression (p trend < 0.0001). Compared to patients in the lowest tertile, those patients with tumors in the highest tertile of TOP2a expression were at increased risk of RCC death (HR=2.31 95% CI 1.64-3.25; p<0.0001). Interestingly, among those patients with low risk disease (SSIGN score 0-3; ∼95% 10 year survival), those with high TOP2a were at increased risk of RCC death (HR=3.09 95% CI 1.29-7.40; p =0.01).

CONCLUSION: Higher TOP2a expression is associated with more aggressive pathologic features and increased risk of cancer-specific death among patients undergoing surgery for localized ccRCC. If confirmed, these data support further inquiry for TOP2a as a prognostic and predictive biomarker for ccRCC patients.

Citation Format: Richard W. Joseph, Dan Serie, Tracy Hilton, Mansi Parasramka, Jeanette Eckel-Passow, John Cheville, Bradley C. Leibovich, Alexander S. Parker. Association of topoisomerase II expression and cancer-specific death in patients with surgically-resected clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 15. doi:10.1158/1538-7445.AM2013-15
Abstract 1501: Pan-cancer PathSeq analysis on TCGA data for pathogen discovery.
Authors: Jung Joonil , Pedamallu Chandra Sekhar , Kostic Aleksander D. , Freeman Samuel , Ojesina Akinyemi I. , Berger Alice , Bhatt Ami , Duke Fujiko , Getz Gad , Meyerson Matthew
Published: August 2014

An estimated 15-20% of all human malignancies is caused by infectious agents, such as human papillomavirus (HPV), Epstein-Barr virus (EBV) or Helicobacter pylori (H. pylori). The recent development of massively parallel next generation sequencing technology led to the discovery of Merkel cell polyomavirus, and more recently a significant enrichment of Fusobacterium nucleatum (F. nucleatum) and related Fusobacterium species in colorectal carcinomas compared to non-neoplastic colon tissue from the same patients. However, these studies involved relatively small number of samples and there are likely many additional cancer-associated microbes that are yet to be discovered. TCGA sequencing data from over 20 human cancer types provide an unprecedented opportunity for cancer-associated pathogen discovery. Using PathSeq, software for pathogen discovery by computational subtraction, we have begun analyzing TCGA sequencing data for the presence of viral, fungal or bacterial sequences. We will present our preliminary data from the analysis of mRNA-seq data including evidence for (1) a greater role of viral integration into tumor suppressor gene loci in HPV-driven tumors and (2) complex interplay between viruses, bacteria and host cancer cells in various cancer types. We will also discuss our discovery efforts for previously unannotated pathogens in the TCGA dataset.

Citation Format: Joonil Jung, Chandra Sekhar Pedamallu, Aleksander D. Kostic, Samuel Freeman, Akinyemi I. Ojesina, Alice Berger, Ami Bhatt, Fujiko Duke, Gad Getz, Matthew Meyerson. Pan-cancer PathSeq analysis on TCGA data for pathogen discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1501. doi:10.1158/1538-7445.AM2013-1501
Abstract 1506: Changes in the expression profile of microRNAs and their predicted targets in fibroblasts associated with breast carcinoma compared to fibroblasts from normal breast tissue.
Authors: Costa Simone V. , Roela Rosimeire A. , Pasini Fatima S. , Katayama Maria L H , Mazzotti Tatiane K. F. , Gebrim Luiz H. , Resende Vanessa C.L. , Neto Miguel S. , Brentani M Mitzi
Published: August 2014

Background: Cancer-associated fibroblasts (CAFs) contribute to tumor growth and progression in breast cancer. CAFs are functional and phenotypically distinct from normal breast tissue (NAFs). Epigenetic modifications and post-transcriptional gene regulation by microRNAs may contribute to the phenotype of CAFs.

Objective: To identify the changes in microRNAs expression profile in fibroblasts associated with breast cancer as compared to those observed in fibroblasts obtained from samples of mammoplasty.

Methodology: Fibroblast primary culture from breast carcinoma (n= 04) and normal breast (n= 06) tissues were established and characterized by Immunohistochemistry. Total RNA extraction and purification were realized using Mirvana kit and miRNAs differentially expressed were identified by qPCR TaqMan® Low Density Array. Potential targets of these miRNAs were identified by in sílico analysis using DIANAm T, Miranda, miRWalk, PICTAR5 and TargetScan databases and compared to microarray profile determined using the U133 plus v.2 Affymetrix chip.

Results: All primary cultures from fibroblast were characterized by presence of vimentin, total absence of CD31 and pan-cytokeratin expression but CAFs exhibited a higher proportion of cells positive for alpha-actin and S100A4. The five microRNAs differentially expressed identified by statistical analysis and grouped hierarchically (hs_miR_655, hs_miR_383, hs_miR_129_3p, hs_miR_15b, hs_miR_489) (FDR≤10% and p<0.05) allowed the separation of the samples into two distinct groups: NAFs and CAFs. The Gene Ontology analysis of predicted target genes to each one of the microRNAs listed above and also identified by microarray, revealed their involvement in important biological processes including cellular morphogenesis and protein localization.

Further detailed studies of these miRNAs and their targets in CAFs may help to better understand the contribution of the mammary tumor microenvironment to breast carcinogenesis and progression.

Financial Support: FAPESP 09/100088-7, FAPESP 2011/08194-3

Citation Format: Simone V. Costa, Rosimeire A. Roela, Fatima S. Pasini, Maria L H Katayama, Tatiane K. F. Mazzotti, Luiz H. Gebrim, Vanessa C.L. Resende, Miguel S. Neto, M Mitzi Brentani. Changes in the expression profile of microRNAs and their predicted targets in fibroblasts associated with breast carcinoma compared to fibroblasts from normal breast tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1506. doi:10.1158/1538-7445.AM2013-1506
Abstract 1516: Melanocyte activation by UVR-induced calprotectin.
Authors: von Maltzan Kristine , Shirley Stephanie H. , Kusewitt Donna F.
Published: August 2014

Melanoma is the most common cause of skin cancer death, although it accounts for only 5% of all skin cancers. Epidemiologic and clinical observations have shown that the ultraviolet (UVR) component of sunlight is the most important etiologic factor in melanoma; however, the precise mechanisms by which UVR drives melanomagenesis are unknown. Mutations in oncogenes and tumor suppressor genes have been identified in melanomas, but most of these are not the UVR signature mutations found in non-melanoma skin cancers. This suggests an indirect role for UVR in melanomagenesis, likely as a tumor promoter. UVR is a potent inducer of keratinocyte-derived paracrine and melanocyte/melanoma cell-derived autocrine factors that profoundly affect melanocyte/melanoma cell gene expression and behavior. Many of these mediators are also potent proinflammatory signals that create an inflammatory environment with additional effects on untransformed and transformed melanocytes. We have identified calprotectin, a secreted heterodimer of S100A8 and A9, as one of these mediators. UVR exposure induces S100A8 and A9 transcription in and calprotectin release from human keratinocytes and melanocytes. Human melanocytes and melanoma cells constitutively express TLR4, one of the receptors for calprotectin. Treatment of human melanocytes and melanoma cells with exogenous calprotectin enhances their proliferation, migration, and invasiveness. UVR-induced calprotectin secretion by melanocytes/melanoma cells and surrounding keratinocytes activates melanocytes/melanoma cells and may thus promote melanomagenesis.

Citation Format: Kristine von Maltzan, Stephanie H. Shirley, Donna F. Kusewitt. Melanocyte activation by UVR-induced calprotectin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1516. doi:10.1158/1538-7445.AM2013-1516
Abstract 1539: Tumor-educated macrophages promote proliferation of human pancreatic cancer cells in vitro and in vivo.
Authors: Hiroshima Yukihiko , Momiyama Masashi , Hassanein Mohamed , Maawy Ali , Menen Rhiana , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M.
Published: August 2014

Macrophages promote tumor growth by stimulating tumor-associated angiogenesis, cancer-cell invasion, migration, and intravasation, as well as suppression of antitumor immune responses. We previously defined the macrophages which were harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as “tumor-educated macrophages (EMϕ)” and demonstrated EMϕ promoted tumor growth and metastasis in orthotopic mouse models. The aim of this study is to elucidate the mechanism of tumor promotion by EMϕ. Transgenic nude mice ubiquitously-expressing GFP were injected subcutaneously with the human pancreatic cancer cell line XPA1 stably expressing RFP in the cytoplasm and GFP in the nucleus. GFP-expressing macrophages from the GFP mice with the dual-color pancreatic tumor were harvested and defined as EMϕ. Macrophages were also harvested from transgenic

GFP mice without tumors and identified as “naïve macrophages (NMϕ)”. EMϕ and NMϕ were compared for their ability to enhance cancer-cell proliferation. Using a skin-flap-window imaging model, the mitotic index of the cancer cells significantly increased after injection of EMϕ compared to NMϕ (p = 0.001). Next, we cultured cancer cells in 3-D on Gelfoam® and found that the cancer cells cultured with EMϕ proliferated to a greater extent compared to those cultured with NMϕ (p = 0.014). We also found that the cancer cells cultured in the conditioned medium harvested from EMϕ had enhanced proliferated compared to cancer cells cultured in the conditioned medium from NMϕ (p = 0.016). These results suggest that EMϕ secrete factors which increase cancer cell growth and can serve as a novel therapeutic target for pancreatic cancer.

Citation Format: Yukihiko Hiroshima, Masashi Momiyama, Mohamed Hassanein, Ali Maawy, Rhiana Menen, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Tumor-educated macrophages promote proliferation of human pancreatic cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1539. doi:10.1158/1538-7445.AM2013-1539
Abstract 1543: Tumor associated macrophages are associated with poor survival in epithelial ovarian cancer.
Authors: El Masri Wafic , Hillerup Caroline , Topka Charlotte , Santiskulvong Chintda , Chiang Alexander , Wu Lily , Rao Jianyu , Dorigo Oliver
Published: August 2014

Background

Tumor associated macrophages have been associated with poor outcomes in several solid tumors, but their impact on the outcome of patients with epithelial ovarian cancer has not been well established.

Methods

We constructed a tissue microarray of 142 epithelial ovarian cancer specimens. Immunohistochemistry was performed for the presence of CD68+ macrophages. The distribution of macrophages was evaluated separately in tumor and stromal tissue. Clinical characteristics were recorded and presence or absence of intratumoral macrophages was correlated with clinical characteristics including survival.

Results

Median follow-up for the entire cohort was 32.0 months (range, 1.7-170 months), and median progression-free survival (PFS) and overall survival (OS) were 14.1 months and 47.8 months, respectively. Intratumoral macrophages (ITMs) were present in 132/142 patients (91.5%) and absent in 12/142 (8.5%). Patients whose tumor tissue did not contain ITMs had a significantly longer median PFS (not reached, p = 0.009) and median OS (150.1 months, p=0.03), compared with the median PFS and OS of 11.7 and 47.7 months, respectively, among patients with ITMs. Interestingly, we could not demonstrate differences in PFS and OS when stromal macrophages were evaluated separately. The presence of ITMs was an independent prognostic factor for OS in a multivariate analysis (p=0.009). The Cox proportional hazard ratios for presence of ITMs were 3.55 for PFS (95% CI, 1.38-12.13) and 3.62 for OS (95% CI, 1.33-13.08).

Conclusions

Our study shows that the presence of intratumoral but not stromal macrophages correlates with poor prognosis in epithelial ovarian carcinoma.

Citation Format: Wafic El Masri, Caroline Hillerup, Charlotte Topka, Chintda Santiskulvong, Alexander Chiang, Lily Wu, Jianyu Rao, Oliver Dorigo. Tumor associated macrophages are associated with poor survival in epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1543. doi:10.1158/1538-7445.AM2013-1543
Abstract 1550: Supernatant of NIH3T3/Src(Y527F) cell line induced Ana-1 macrophages to tumor-associated macrophages by IL-6.
Authors: Chen Lechuang , Hu Chenfei , Xu Qing , Ma Kai , Liu Mei , Zhu Hongxia , Xu Ningzhi
Published: August 2014

Macrophages (Mф) is the major components of the solid tumors. Tumor-associated macrophages (TAM) play an important role in the development of tumor inflammation. The tumor microenvironment could induce macrophages differentiation into TAM, and the TAM could regulate tumor growth and invasion.

However, the mechanisms that by which signaling pathways contribute to cell differentiation are not well deﬁned. As we known, the tumor microenvironment in vivo is complex and integrated by a variety of cells and factors. In this study, we chose NIH3T3 cell line to construct a stable Src(Y527F) expression system. We found that its supernatant could induce macrophages differentiation into TAM in vitro. When we injected the cell mixture of Ana-1 macrophages and NIH3T3/Src(Y527F) cells into nude mice, the volume of tumors increased more quickly than the control group that just injected NIH3T3/Src(Y527F) cells only. And, we also found that the tumors became more invasive than control tumors. Furthermore, 62 cytokines were tested by using Cytokine Antibody Array, the result showed that 13 of them significantly changed in the supernatant of NIH3T3/Src(Y527F) cells compared with NIH3T3. Strikingly, IL-6 was 71-fold up-regulated. When we added IL-6 in the supernatant, it could also induce Ana-1 macrophages differentiation into a TAM phenotype. Our findings indicated that IL-6 might play an important role in the differentiation of Ana-1 from macrophages to TAM by using the supernatant of NIH3T3/Src(Y527F) cell line.

Citation Format: Lechuang Chen, Chenfei Hu, Qing Xu, Kai Ma, Mei Liu, Hongxia Zhu, Ningzhi Xu. Supernatant of NIH3T3/Src(Y527F) cell line induced Ana-1 macrophages to tumor-associated macrophages by IL-6. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1550. doi:10.1158/1538-7445.AM2013-1550
Abstract 1556: Immunological naturalization of immunocompetent host mice to luciferase-GFP for consistent tracking of transplanted tumors.
Authors: Day Chi-Ping , Weaver Zoe , Carter John , Bonomi Carrie , Van Dyke Terry , Hollingshead Melinda , Merlino Glenn
Published: August 2014

Optical imaging of labeled cells is the most prevailing method for cell tracking in mouse models. However, the immunogenicity of xenobiotic reporter genes, such as the commonly used firefly luciferase (ffLuc) and enhanced green fluorescence protein (eGFP), results in inconsistency in cell labeling and tumor progression, preventing their use in host mice with normal immunity. To resolve this issue, we have generated a reporter gene-tolerized transgenic mouse in which an ffLuc-eGFP fusion gene was targeted to the anterior pituitary gland using a rat Growth Hormone promoter (dubbed the “glowing-head” mouse). When ffLuc-eGFP-labeled Lewis Lung Carcinoma (LLC) tissue was subcutaneously transplanted into syngeneic glowing-head mice (GH-c-brd), their non-transgenic litter-mates (WT-c-brd), and immunocompromised Nod-Scid (balb/c) mice, anti-GFP antibody was induced only in the circulation of WT-c-brd. Labeled LLC also exhibited fewer CD4+ T cells but more macrophages in GH-c-brd than in WT-c-brd, suggesting it induced adaptive immunity in the latter. Moreover, GH-c-brd exhibited faster subcutaneous tumor growth and post-resection metastatic progression, and maintained more consistent tumor labeling, as compared to WT-c-brd and even Nod-Scid mice. Interestingly, in an adjuvant chemotherapeutic setting, the immunogenicity of the labeled tumor was able to deter metastatic progression in a manner similar to that obtained by drug treatment. We conclude that the immunogenicity of xenobiotic labeling markers can significantly influence disease progression and therapeutic responses thereby compromising immunocompetent preclinical cancer models, and that the glowing-head mouse can be used to circumvent such problems and promote consistent disease monitoring.

Citation Format: Chi-Ping Day, Zoe Weaver, John Carter, Carrie Bonomi, Terry Van Dyke, Melinda Hollingshead, Glenn Merlino. Immunological naturalization of immunocompetent host mice to luciferase-GFP for consistent tracking of transplanted tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1556. doi:10.1158/1538-7445.AM2013-1556
Abstract 1567: Use of humanized NOG mice within the pre-clinical development of new human specific therapies.
Authors: Mignard Caroline , Mirjolet Jean-Francois , Kissel Holger , Bichat Francis , Duchamp Olivier
Published: August 2014

Immune cells within the tumor microenvironment are now well recognized as to be targets of interest for cancer treatment. However there are no available pre-clinical models that will accurately support the pre-clinical development of such therapeutic approaches. Humanized NOG (NOD/shi-scid/IL-2Rγnull) mice, bearing human immune cells with or without human target tumor cells, are relevant models to test various therapeutic strategies (e.g. antibody dependent cell cytotoxicity, Treg targeting antibodies, TLR agonists, vaccines, adoptive T cells transfer, …) in various pathologies (e.g. oncology, autoimmune disease, inflammation, transplantation, …). Humanization of NOG mice was already characterized and validated using freshly collected human PBMCs or hematopoietic stem cells (HSCs) in both newborn and adult NOG mice.

The complete characterization of the reconstituted NOG mice was done in peripheral blood as well as central lymphoid organs such as bone marrow, spleen and thymus, using FACS analysis. Moreover, the human immune reconstitution and function in NOG mice was also evidenced with human cytokine and immunoglobulin quantification in mouse plasma samples. The injection of mature human PBMC in adult NOG mice leads to the consistent development of a xenogeneic graft-versus-host disease (GvHD), which mimics human GvHD (modification of CD4/CD8 ratio, expression of T cells activation markers and cytokine production).

Growth characteristics of human tumors models on humanized mice will be presented either as subcutaneous or as disseminated intravenous model. Randomization parameters were selected regarding both tumor and immune cell sides. Required readouts to understand immune cell modulation (Cell phenotyping and functionality) and related antitumor efficacy will be described. Finally, such models, i.e. humanized NOG mice bearing or not human tumor cells in the context of evaluation of new therapies will be detailed in various pathologies.

Citation Format: Caroline Mignard, Jean-Francois Mirjolet, Holger Kissel, Francis Bichat, Olivier Duchamp. Use of humanized NOG mice within the pre-clinical development of new human specific therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1567. doi:10.1158/1538-7445.AM2013-1567
Abstract 1577: Subtype dependent carcinoma initiation by Rb tumor suppressor inactivation in multiple epithelial tissues.
Authors: Song Yurong , Yang Chunyu , Pan Wenqi , Fathalizadeh Alisan , Lu Lucy , Gilbert Debra , O'Sullivan T. Norene , Haines Diana C. , Martin Philip L. , Van Dyke Terry
Published: August 2014

Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by mesenchymal cells. Of major importance is to understand the relationship among cell types, initiation event, and ultimate cancer outcome. Mouse models facilitate the prospective examination of early oncogenic events, which is not feasible in humans, permitting an analysis of epithelial subtype susceptibility. Since most solid tumors harbor aberrations in the pRb network, we assessed the susceptibility of epithelial subtypes expressing cytokeratin (K) 18 or K19 to the disruption of

Rb tumor suppression (Rb-TS) in vivo. Tumorigenesis could be initiated in either K18- or

K19-expressing cells. However, the susceptibility of epithelial tissues was subtype-dependent. K19 cells were more prone to hyperplasia and neoplasia than

K18 targeted cells. Given that a single genetic event was engineered, a small percentage of animals developed carcinoma/adenocarcinoma, providing the opportunity to define progression events via engineering or spontaneous evolution. To extend our analysis to a specific tissue, we employed a prostate-specific Cre line to target either K18- or K19-expressing prostate epithelial cells. Differential responses observed in the frequency and extent of premalignant hyperplastic lesions strongly supported the notion of cell type-dependent susceptibility to Rb-TS inactivation. Our data suggest that the tumorigenic consequences of a single initiation event are dictated by cellular subtype distinctiveness, underscoring the importance of the tumor cell of origin.

Citation Format: Yurong Song, Chunyu Yang, Wenqi Pan, Alisan Fathalizadeh, Lucy Lu, Debra Gilbert, T. Norene O'Sullivan, Diana C. Haines, Philip L. Martin, Terry Van Dyke. Subtype dependent carcinoma initiation by Rb tumor suppressor inactivation in multiple epithelial tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1577. doi:10.1158/1538-7445.AM2013-1577
Abstract 1579: Establishment of transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs.
Authors: Matsuzaki Yuriko , Hosokai Haru , Mizuguchi Yukiyo , Shimizu Atsushi , Saya Hideyuki
Published: August 2014

The development of anticancer drugs would be facilitated by the availability of an in vivo screening system based on a multicellular organism. We intended to construct an animal model which can be stable and easily detected tumors without autopsy for chemical screening and assessment. In this study, the medaka which is a small egg-laying freshwater fish native to East Asia was applied.

The medaka has proved suitable for toxicology and carcinogenesis in the past studies. Furthermore most of molecular technology is available to the medaka and the size of the medaka genome (∼800 Mb) is about half that of the zebrafish genome. To construct a tumor model using transgenic medaka fish, a promoter sequence suitable for the tissue specific tumorigenesis was selected. There are some genes having specific expression in melanocyte. Melanocyte specific expression of oncogenes may induce progression to melanoma. Therefore we selected a promoter sequence encoding Tyrosinase, one of the melanocyte specific genes.

The recombinant plasmids for microinjection are consisting of the medaka tyrosinase promoter and human oncogenes (cMyc, BRAF mutant V600E and HRAS mutant G12V). One cell stage embryos were applied for microinjection. We obtained three stable transgenic individuals derived from one cMyc and two BRAF mutant (BRAF600E) injected F0, but none from HRAS mutant (HRAS12V). The transgenic (Tg)-cMyc and the Tg-BRAF600E produced tumor in internal organs with more than 50 % of all fish, but in eyes or skins (outer detectable organs) with less than 10 %. The Tg-HRAS12V animals died before achieving adulthood. We therefore developed a conditional gene expression system in which HRAS12V is expressed in response to the induction of Cre recombinase by heat treatment, given that the Cre gene was placed under the control of a medaka heat shock promoter.

One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. This Tg-HRAS12V medaka established here thus represents a promising in vivo system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes.

Citation Format: Yuriko Matsuzaki, Haru Hosokai, Yukiyo Mizuguchi, Atsushi Shimizu, Hideyuki Saya. Establishment of transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2013-1579