bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4863: Prostaglandin and associated eicosanoid dose response to omega-3 fatty acid (FA) diet in rat colon.
Authors: Jiang Yan , Djuric Zora , Ren Jianwei , Dmitry Kuklev V. , Curtis Austin , Bentz Gretchen S. , Waters Ian , Sen Ananda , Zhao Lili , Uhlson Charis , Murphy Robert , Smith William L. , Brenner Dean E.
Published: August 2014

Introduction: Prostaglandin (PG) E2 has been linked to higher risk of colorectal carcinogenesis. Our previous in vitro data (Wada et al. J Biol Chem 2007;282;22254) demonstrated reduction of cellular eicosanoids by substituting the n-3 fatty acid EPA for the more common n-6 FA arachidonic acid (AA) as substrate for the cyclooxygenases (Cox). We tested the hypothesis that EPA substitution for n-6 FA in vivo will reduce procarcinogenic eicosanoids in rats.

Methods: F344 male rats were fed EPA: n-6 FA ratio diets of 0, 0.1, 0.2, 0.4, 0.6 for 5 weeks. Eicosanoids (PGs and lipoxins) in proximal, transverse and distal colonic tissue and urinary PG metabolites were assayed on LC-MS-MS. Fatty acids in serum and tissue were measured by GC-MS.

Results: Serum EPA:AA ratio (means(SD)) increased linearly from 0.02(0.004) at baseline (no EPA) to 0.98(0.32) EPA:n-6 ratio of 0.6. Colonic EPA:AA ratios increased from 0.03(0.01) to 0.41(0.15) in distal colon. Representative eicosanoid concentrations (μg/mg protein) in distal colon tissue with associated EPA:n-6 diets in the Table suggest rapid eicosanoid reduction at lower dietary EPA:n-6 ratios and then flattening of the dose response at higher ratios. The distal colon contained the lowest eicosanoid concentrations compared to transverse and proximal colon. Urinary PGE metabolite was reduced from 13.42(1.95) ng/mg creatinine (dietary EPA:n-6 FA=0) to 7.67(0.62) ng/mg creatinine (dietary EPA:n-6 FA=0.6).

Conclusion: Diet induced substitution of omega-3 for omega-6 substrates of Cox and lipoxygenases in F-344 rats causes a rapid, non-linear decline in colonic PGE2 and lipoxin products. PGE3 increases linearly with omega-3 FA dose. These data support the hypothesis that reduction in carcinogenesis stress induced by eicosanoids can be modulated by a dietary intervention in vivo.

Eicosanoids in rat colon EPA: n-6 ratio in diet PGE2 PGE3 5-HETE 12-HETE 0 264.43(77.54) 0.12 (0.14) 2.28 (1.74) 35.67 (26.95) 0.2 116.56 (50.53) 0.89 (0.81) 1.15 (0.99) 11.73 (7.65) 0.4 86.15 (27.28) 1.52 (0.96) 1.80 (2.02) 5.93 (4.01) 0.6 90.89 (20.90) 2.17 (1.36) 1.52 (0.78) 5.68 (2.77)

Citation Format: Yan Jiang, Zora Djuric, Jianwei Ren, Kuklev V. Dmitry, Austin Curtis, Gretchen S. Bentz, Ian Waters, Ananda Sen, Lili Zhao, Charis Uhlson, Robert Murphy, William L. Smith, Dean E. Brenner. Prostaglandin and associated eicosanoid dose response to omega-3 fatty acid (FA) diet in rat colon. [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 4863. doi:10.1158/1538-7445.AM2013-4863
Abstract 4870: High dietary vitamin D prevents dysplastic lesions in an AOM/DSS model of colorectal cancer.
Authors: Hummel Doris M. , Höbaus Julia , Thiem Ursula , Graca Joao , Mesteri Ildiko , Gober Lukas , Obermayer-Pietsch Barbara , Kallay Enikö
Published: August 2014

Diet and lifestyle have an important impact on the etiology of colorectal cancer (CRC), one of the leading causes of cancer morbidity and mortality in Western countries. Epidemiological studies suggest that vitamin D might have chemopreventive effects.

In this study we investigated the effect of increasing dietary vitamin D3 intake on chemically induced colorectal tumor development in mice.

We fed female C57BL/6J mice with a diet containing five different vitamin D3 concentrations ranging from 100 to 5000 IU/kg diet and studied the impact of dietary vitamin D3 on the development of chemically induced colonic dysplasia, a precursor of CRC. To induce dysplasia, we injected mice once with 10 mg/kg azoxymethane (AOM) intraperitoneally, followed by three cycles of dextran sodium sulfate salt (DSS) in the drinking water. Dietary vitamin D3 concentration correlated positively with 25(OH)D3 serum levels (Spearman Correlation Coefficient (SCC) 0.752, p<0.001). Both dietary vitamin D3 concentration and serum 25-hydroxyvitamin D3 levels correlated inversely with the dysplasia score (SCC -0.579, p=0.002; SCC -0.618, p=0.001). Dysplastic regions expressed less vitamin D receptor while the proliferation marker Ki67 was significantly higher expressed in regions with high grade dysplasia compared with the respective normal mucosa of the same colon.

Our data show that high dietary vitamin D3 concentrations reduce the development of chemically induced precursors of colorectal cancer in mice.

Citation Format: Doris M. Hummel, Julia Höbaus, Ursula Thiem, Joao Graca, Ildiko Mesteri, Lukas Gober, Barbara Obermayer-Pietsch, Enikö Kallay. High dietary vitamin D prevents dysplastic lesions in an AOM/DSS model of colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4870. doi:10.1158/1538-7445.AM2013-4870
Abstract 4901: BMI1 is not a marker of cancer or stem cells in the prostate.
Authors: Pellakuru Laxmi Gurunatham , Ocal-Gumuskaya Berrak , Zheng Qizhi , Hicks Jessica , De Marzo Angelo
Published: August 2014

The polycomb gene, BMI1, has been reported to be critical in regulating self-renewal and proliferation of a number of tissue-specific normal as well as cancer stem cells, and it has been shown to be overexpressed in several tumor types. BMI1 has been reported to be highly expressed in the prostate basal cell compartment (Lukacs et al 2010, Cell Stem Cell 7: 682-693), which houses prostate stem cells (Robinson et al 1998, Prostate 37: 149-160), as well as being upregulated in prostate cancer (Lukacs et al 2010, van Leenders et al 2007, Eur Urol 52: 455-463). However, other studies have not found BMI1 levels to correlate with prostate cancer (Wolters et al 2010, BJU Int 106: 280-286). We used immunohistochemical staining as well as chromogenic in situ hybridization to characterize the pattern of BMI1 expression in normal prostate as well as primary and metastatic adenocarcinoma specimens using both standard glass slides and tissue microarrays. For our IHC staining, we used the antibody from Cell Signaling, and we validated that on human prostate tissue, a single band is obtained by western immunoblot. Using this antibody, we were unable to show any consistent difference in BMI1 protein between prostate basal and luminal cells, with generally abundant staining in both compartments. Similar findings were seen with in situ hybridization for BMI1 mRNA. We also found that BMI1 is not consistently upregulated in primary or metastatic human prostate cancers, albeit most tumor cells in most cases showed robust nuclear staining. The findings that BMI1 is not enriched in the stem cell compartment, does not label only a subset of prostate cancer cells, and is not generally upregulated in prostate cancer to any significant degree supports the concept that BMI1 would not be considered a marker of stem cells, cancer stem cells or a cancer biomarker in the prostate.

Citation Format: Laxmi Gurunatham Pellakuru, Berrak Ocal-Gumuskaya, Qizhi Zheng, Jessica Hicks, Angelo De Marzo. BMI1 is not a marker of cancer or stem cells in the prostate. [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 4901. doi:10.1158/1538-7445.AM2013-4901
Abstract 4904: Activation of aldehyde dehydrogenase is essential for growth of mesenchymal glioma stem cells.
Authors: Mao Ping , Joshi Kaushal , Li Jian feng , Godlewski Jakub , Mo Xiao kui , Chen Shi yuan , Sobol Robert W. , Nakano Ichiro
Published: August 2014

Emerging evidence has indicated glioma stem cells(GSCs) could be subclassified into two major entities termed as as proneural(PN) GSCs and mesenchymal(MES) GSCs according to their mutually-exclusive gene expression pattern. In this study, we characterize 20 patient-derived GSCs into PN GSCs and MES GSCs based on the unbiased analysis with transcriptome microarray and microRNA profiles. Signaling pathway analysis identified the upregulation of various tumor metabolic pathways in MES GSCs. In particular, the glycolytic pathway containing several aldehyde dehydrogenase (ALDH) genes was significantly enriched in MES GSCs. ALDH activity was elevated in MES GSCs, but not in MES non-GSCs or PN GSCs, and its inhibition attenuated MES GSC growth. Intriguingly, irradiation could induce PN GSC's loss of PN phenotype and a gain of MES phenotype, indicating the irradiation-induced PN-to-MES transformation. Furthermore, selective ALDH inhibitor, DEAB, can partially block irradiation-induced transformation of PN to MES GSCs. Clinically, ALDH immunoreactivity was markedly elevated in malignant gliomas in comparison to low grade gliomas or normal brain tissues. Collectively, our data suggest that two GSC subtypes harboring distinct signaling pathways represent intertumoral glioma heterogeneity, and highlight previously unidentified roles of ALDH-associated metabolic signaling in MES MGs and their GSCs. Blocking the pathways that regulate ALDH activity might providepromising therapeutic approaches for a subset of malignant gliomas with the MES signature.

Citation Format: Ping Mao, Kaushal Joshi, Jian feng Li, Jakub Godlewski, Xiao kui Mo, Shi yuan Chen, Robert W. Sobol, Ichiro Nakano. Activation of aldehyde dehydrogenase is essential for growth of mesenchymal glioma stem 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 4904. doi:10.1158/1538-7445.AM2013-4904
Abstract 4907: Knockdown of MMP-9 inhibits radiation-induced invasive phenotype, angiogenesis and tumor growth in glioma cancer stem cells.
Authors: Chetty Chandramu , Ponnala Shivani , Gujrati Meena , Rao Jasti S.
Published: August 2014

Gliomas contain a small number of treatment-resistant glioma stem cells (GSCs). It is thought that tumor regrowth or recurrence originates from GSCs, thus rendering GSCs an attractive target for novel treatment approaches. GSCs are also resistant to conventional chemotherapy and radiation therapy, and a low dose of ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. In the present study, an isolated glioma stem cell population exhibited a significant differential expression of MMP-9 and a low dose of irradiation enhanced MMP-9 expression levels. Transfection of GSCs with pMMP-9-Si inhibited radiation-induced MMP-9 activity and protein levels as well as cell proliferation. The sphere de-differentiating and invasive ability of GSCs were also reduced in MMP-9 knockdown cells when compared with mock and pSV controls. MMP-9 knockdown led to the induction of apoptotic cell death as determined by FACS and TUNEL analyses, and caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage. pMMP-9-Si transfection also inhibited radiation-induced angiogenesis in vitro and in vivo in GSCs. Further, pMMP-9-Si treatment inhibited pre-established glioma tumor growth in an intracranial model. In addition, GSC xenograft tissue sections from mice that were treated with MMP-9 siRNA showed reduced expression of VEGF and the angiogenic marker, CD31. Indeed, TUNEL analysis of tumor sections collected from the combination group of pMMP-9-Si and irradiation revealed more apoptotic cells. These results validate the usefulness of targeting MMP-9 and provide a novel perspective in the treatment of the cancer stem cell population with its higher resistance to radiotherapy.

Citation Format: Chandramu Chetty, Shivani Ponnala, Meena Gujrati, Jasti S. Rao. Knockdown of MMP-9 inhibits radiation-induced invasive phenotype, angiogenesis and tumor growth in glioma cancer stem 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 4907. doi:10.1158/1538-7445.AM2013-4907
Abstract 4910: The search for the stem cells of human and canine osteosarcoma: evaluation of stemness markers and sarcosphere formation.
Authors: Yang Ya-Ting , Yuzbasiyan-Gurkan Vilma
Published: August 2014

Osteosarcoma (OS) is the most common primary bone tumor of both humans and dogs. The high recurrence rate, resistance to chemotherapy and radiotherapy indicate that novel approaches to treatment are needed. In various cancers, a small population of cells with capacity for self-renewal, tumor growth and metastasis have been identified and designated cancer stem cells, CSCs. Identification and effective targeting of CSCs are necessary for successful treatment of these cancers. The heterogeneous cell types of OS suggest the involvement of CSCs in OS. In some studies, the ability to form spheres in semi-solid media has been proposed as a characteristic of CSCs and in others, the expression of the general stemness markers such as OCT4 has been proposed as a major phenotype of the CSCs. In this study, we evaluated the sarcosphere forming fraction of two cell lines, D17 and SAOS2, a canine and a human OS cell line, respectively. Both D17 and SAOS2 cells were transfected with an OCT4-YFP (yellow florescent protein) reporter construct. The YFP positive and negative cell fractions were isolated from both cell lines and evaluated for sarcosphere forming ability. In addition, expression of OCT4 and other stemness markers, NANOG and SOX2 were evaluated. All cells were characterized for their tumorigenic potential in vitro, including invasion through Matrigel, as well as for their ability to form sarcospheres. Our data show that OCT4 positive cells were more invasive than negative subsets in both D17 and SAOS2 cell lines. In addition, while both subsets were able to form spheres, OCT4 positive cells formed larger spheres than negative cells. These findings further highlight the heterogeneity of the osteosarcoma tumor cells, and suggest that neither sarcosphere formation nor OCT4 expression are sufficient to define the CSCs in either canine and human OS. Further studies incorporating other markers are ongoing to identify and characterize the CSCs in OS.

Citation Format: Ya-Ting Yang, Vilma Yuzbasiyan-Gurkan. The search for the stem cells of human and canine osteosarcoma: evaluation of stemness markers and sarcosphere formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4910. doi:10.1158/1538-7445.AM2013-4910
Abstract 4912: MELK-dependent phosphorylation of FOXM1 is essential for mitotic progression of glioma stem cells.
Authors: Joshi Kaushal , Nakano Ichiro
Published: August 2014

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator for mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the serine/threonine kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. The MELK-dependent activation of FOXM1 results in an increase of mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1 (Polo-like kinase 1). Using neural progenitor cells (NPCs) derived from mouse brains, we found that transgene expression of FOXM1 enhances, while its siRNA-mediated gene silencing diminishes clonal neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases from NPCs, pre-tumorigenic progenitors, and GSCs. As expected, sensitivity of GSCs to pharmacological inhibition of MELK-mediated FOXM1 signaling via the thiazole antibiotic Siomycin A is substantially higher than NPCs. In addition, treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cell culture, and addition of Siomycin A to Temozolomide treatment to mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with the kinase domain of MELK regulates mitotic key genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.

Citation Format: Kaushal Joshi, Ichiro Nakano. MELK-dependent phosphorylation of FOXM1 is essential for mitotic progression of glioma stem 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 4912. doi:10.1158/1538-7445.AM2013-4912
Abstract 4917: CD44 standard form expression in human ovarian carcinoma is correlated with disease progression but not prognosis.
Authors: Zhang Jing , Chang Bin , Liu Jinsong
Published: August 2014

Single-chain glycoprotein CD44 is a major cell surface receptor for hyaluronan and mediates epithelial cell adhesion by its involvement in cell-cell and cell-matrix interactions. Recently, CD44 has been identified as a biomarker of cancer stem cells in many malignancies, including ovarian carcinoma. However, its clinical significance in human ovarian carcinoma has been controversial until recently. The aim of our current study was to clarify the clinical role of CD44 expression in ovarian carcinoma. Immunohistochemical staining of 483 human primary ovarian carcinoma, and 27 paired primary and recurrent ovarian carcinoma samples for CD44 standard form (CD44s) was performed using tissue microarray. The associations between CD44s expression and clinical factors (diagnosis, tumor grade, International Federation of Gynecology and Obstetrics stage, and response to chemotherapy) and overall and disease-free survivals were analyzed. We observed CD44s expression in 38.1% of the ovarian carcinoma samples. Results of Fisher's exact test suggested that CD44s expression was associated with high-grade carcinoma (P = 0.013), especially high-grade serous carcinoma (P = 0.037); International Federation of Gynecology and Obstetrics stage (III-IV, P < 0.001); age at diagnosis less than 60 years old (P = 0.011); and transitional cell carcinoma (P = 0.039). However, CD44s expression was not associated with overall survival (P = 0.529) or disease-free survival (P = 0.035) by the log-rank test. Moreover, there was no statistical difference in CD44s expression between the primary and recurrent epithelial ovarian carcinomas. Our results showed that CD44s expression is correlated with tumor progression but not prognosis.

Citation Format: Jing Zhang, Bin Chang, Jinsong Liu. CD44 standard form expression in human ovarian carcinoma is correlated with disease progression but not prognosis. [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 4917. doi:10.1158/1538-7445.AM2013-4917
Abstract 4936: Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop.
Authors: Hayes Karen E. , Walk Elyse L. , Ammer Amanda G. , Kelley Laura C. , Martin Karen H. , Weed Scott A.
Published: August 2014

Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src transformed Abl-/-/Arg-/- fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with therapeutic inhibitors of the EGFR invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating an EGFR-Src-cortactin cascade, in contrast to the invasion promoting functions of Abl kinases in breast and other cancer types. Our results provide mechanistic support for recent failed HNSCC clinical trials utilizing imatinib.

Citation Format: Karen E. Hayes, Elyse L. Walk, Amanda G. Ammer, Laura C. Kelley, Karen H. Martin, Scott A. Weed. Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop. [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 4936. doi:10.1158/1538-7445.AM2013-4936
Abstract 4939: MicroRNA-375 regulates tumor invasion and metastasis phenotypes and is associated with poor outcome in head and neck squamous cell carcinoma.
Authors: Jimenez Lizandra , Harris Thomas , Kawachi Nicole , Belbin Thomas , Schlecht Nicolas , Lim Jihyeon , Angeletti Ruth , Prystowsky Michael B. , Childs Geoffrey , Segall Jeffrey
Published: August 2014

Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer, with a five-year survival rate of around 50%. Our goal is to improve patient outcome through understanding the mechanisms determining HNSCC invasiveness. Our research group identified miR-375 as the most consistently down-regulated miRNA in tumor samples when compared to paired normal samples. Patients in the lowest quartile of miR-375 expression had significantly decreased disease-specific survival, increase incidence of loco-regional recurrence and distant metastasis. The highly significant association of lower miR-375 expression in patients with poor prognosis and distant metastasis led us to investigate the mechanism by which miR-375 expression causes alterations to the invasive properties of HNSCC. Stable transductant cell lines with increased expression of miR-375 have been generated from two oral cavity cell lines, UMSCC1 and UMSCC47, utilizing lentiviral constructs driving expression of the miR-375 precursor. We have not seen a significant change in proliferation rate for either of the miR-375 expressing lines compared to the empty vector control expressing lines. We have observed diminished invasion properties in vitro correlates with increased miR-375 expression. We have also identified that miR-375 expression in HNSCC result in impaired chemotactic migration properties and suppressed matrix degradation properties. To identify proteins regulated by miR-375, we are utilizing stable isotope labeling of cells in cell culture (SILAC) to identify candidate miRNA target proteins to identify differentially expressed proteins in control and miR-375 over-expressing lines. Candidate target proteins will be validated as targets of miR-375 and tested for involvement in the diminished invasive properties of HNSCC as a result of miR-375 expression. In conclusion, miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and may function by suppressing the expression of genes and proteins which are involved in the invasive properties of HNSCC.

Citation Format: Lizandra Jimenez, Thomas Harris, Nicole Kawachi, Thomas Belbin, Nicolas Schlecht, Jihyeon Lim, Ruth Angeletti, Michael B. Prystowsky, Geoffrey Childs, Jeffrey Segall. MicroRNA-375 regulates tumor invasion and metastasis phenotypes and is associated with poor outcome in head and neck squamous 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 4939. doi:10.1158/1538-7445.AM2013-4939
Abstract 495: Immune response to XAGE-1b (GAGED2a) in NSCLC patients and its clinical relevance.
Authors: Ohue Yoshihiro , Kurose Koji , Eikawa Shingo , Mizote Yu , Matsumoto Hirofumi , Isobe Midori , Uenaka Akiko , Fukuda Minoru , Nakayama Eiichi , Oka Mikio
Published: August 2014

BACKGROUND:XAGE-1bis a cancer/testis (CT) antigen expressed highly in non-small cell lung cancer (NSCLC) with restricted expressiononly in testis in normal tissues. In this study, we investigated correlation of intensity of humoral and cellular immune responses against XAGE-1b and itsclinical relevance in NSCLC patients.

MATERIALS AND METHODS:Peripheral bloodwas obtained from NSCLC patients who visited Kawasaki Medical School Hospital between 2005 and 2012 under written informed consent. Antibody response to XAGE-1bwas analyzed by ELISA using synthesized XAGE-1b protein. CD4 and CD8 T-cell responses were examined by IFN-γ ELISA and/or IFN-γcapture assay by FACS using 12-mer or 16-merXAGE-1b-overlapping peptides (OLPs) spanning the entire XAGE-1b (GAGED2a) protein.

RESULTS: Antibody positive frequencies of total 362 NSCLC, 220 lung adenocarcinoma and 85 lung squamous cell carcinoma patients were 8.8% (32/362), 12.7% (28/220) and 1.2% (1/85), respectively. With antibody positive NSCLC patients, the patients showing high, intermediate and low antibody response was 7, 20 and 5, respectively. The frequency of XAGE-1b-reactive CD4 T-cells in patients showing high, intermediate and low antibody response was 5.5 ±2.1 x 10−5, 1.5 ±0.7 x 10−5 and < 1.1 x 10−5, respectively. The frequency of XAGE-1b-reactive CD8 T-cells was 6.9 ±1.6 x 10−6, 4.6 ±1.6 x 10−6 and < 1.1 x 10−6, respectively. The frequency of cytotoxic CD8 T-cells in IFN-γ secreting CD8 T-cells in response to XAGE-1b-peptide-pulsed autologous EBV-B cells in patients showing high and intermediate antibody responses was 59.0% and 4.8%, respectively.We evaluated overall survival (OS) time with 120 stage IIIB/IV lung adenocarcinoma patients. Median OS were 32.3 and 14.4 months in antibody-positive and negative patients(P-.039). There was no significant differencewith 1-year survival rate in antibody-positive (68.7%) and negative (55.6%)patients, but was significant with 3-year survival rate in 34.8% and 14.1% respectively. Furthermore, univariate and multivariable analyses showed XAGE-1b antibody response was independent prognostic factor.

CONCLUSION:Our findings indicate that CT antigen XAGE-1b is highly immunogenic in NSCLC patients inducing antibody, and CD4 and CD8 T-cell responses and the immune response against XAGE-1b is beneficial for prognosis.

Citation Format: Yoshihiro Ohue, Koji Kurose, Shingo Eikawa, Yu Mizote, Hirofumi Matsumoto, Midori Isobe, Akiko Uenaka, Minoru Fukuda, Eiichi Nakayama, Mikio Oka. Immune response to XAGE-1b (GAGED2a) in NSCLC patients and its clinical relevance. [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 495. doi:10.1158/1538-7445.AM2013-495
Abstract 4953: Stromal endosialin modulates the proinflammatory tumor microenvironment and is crucial for the growth of orthotopic pancreatic tumors.
Authors: Christian Sven , Sivanandam Vijaysahankar , Strerath Michael , Apeler Heiner , Hess-Stumpp Holger , Augustin Hellmut G.
Published: August 2014

Endosialin (CD248), a transmembrane protein expressed in stromal fibroblasts and activated pericytes during embryonic development, is expressed in human carcinomas. Although its function is unknown, due to its highly specific expression pattern endosialin represents a promising anti-tumor target. To unravel the role of endosialin during tumor progression and metastasis, we employed systematic loss-of-function approaches in stromal fibroblasts in vitro as well as in subcutaneous and orthotopic tumors in vivo. These experiments revealed an important role of endosialin in regulating fibroblast proliferation and in orchestrating an inflammatory response by modulating the secretion of inflammatory cytokines (IL6) in stromal fibroblasts. The proinflammatory environment created by activated fibroblasts is thought to support tumor growth and metastasis in vivo. Experiments in endosialin-deficient mice confirmed an important role of endosialin in tumor progression in highly stroma-recruiting orthotopic pancreatic tumor models. Finally, lung metastases were dramatically decreased in endosialin-deficient mice supporting a role of a proinflammatory tumor stroma in tumor dissemination. Taken together, our experiments identify endosialin as an important modulator of stromal responses that are crucial for tumor progression and metastasis. Endosialin may therefore represent a novel target for development of anticancer therapies for solid tumors.

Citation Format: Sven Christian, Vijaysahankar Sivanandam, Michael Strerath, Heiner Apeler, Holger Hess-Stumpp, Hellmut G. Augustin. Stromal endosialin modulates the proinflammatory tumor microenvironment and is crucial for the growth of orthotopic pancreatic 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 4953. doi:10.1158/1538-7445.AM2013-4953
Abstract 4959: Targeting pancreatic stellate cells (PSC) to treat pancreatic cancer.
Authors: Gonzalez-Villasana Vianey , Rodriguez-Aguayo Cristian , Arumugam Thiru , Cruz-Monserrate Zobeida , Deng Defeng , Fuentes-Mattei Enrique , Hwang Rosa F. , Wang Huamin , Pena Guillermo N. Armaiz , Aslan Burcu , Gutierrez-Puente Yolanda , Sood Anil K. , Logsdon Craig , Lopez-Berestein Gabriel
Published: August 2014

Pancreatic cancer (PC) is characterized by a prominent stromal reaction. Recently, the contribution of the stromal elements to the initiation and progression of the disease has come to be appreciated. In this context, targeting this tumor microenvironment represents a potential strategy to improve the delivery and efficacy of chemotherapeutic agents leading a better prognosis for the patients. Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosis in pancreatic cancer. In the current study, we observed that PSC possess α-naphthyl acetate esterase (ANAE) activity, which is a specific marker enzyme for monocytes and macrophages, these cells can be differentiated/activated in a similar fashion as a monocytes/macrophages, suggesting that PSC behave in a similar fashion as macrophages, and hence can be treated as such. Based on these data we targeted PSC in vitro and in vivo by using bisphosphonates (BPs) that are macrophages inhibitors. BPs inhibited proliferation and inactivated PSC in vitro through inhibition of protein prenylation, and increased cell apoptosis and cell cycle arrest in G1. Furthermore, BPs inactivated PSC, inhibited tumor growth (p<0.0005), tumor weight (p<0.001) and number of nodules (p<0.001) in an orthotopic animal model of pancreas. These antitumor effects were enhanced when BPs were combined with a taxane. These data suggest that BPs could disrupt the pancreatic cancer, which could offer a better prognosis to PC patients.

Citation Format: Vianey Gonzalez-Villasana, Cristian Rodriguez-Aguayo, Thiru Arumugam, Zobeida Cruz-Monserrate, Defeng Deng, Enrique Fuentes-Mattei, Rosa F. Hwang, Huamin Wang, Guillermo N. Armaiz Pena, Burcu Aslan, Yolanda Gutierrez-Puente, Anil K. Sood, Craig Logsdon, Gabriel Lopez-Berestein. Targeting pancreatic stellate cells (PSC) to treat pancreatic 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 4959. doi:10.1158/1538-7445.AM2013-4959
Abstract 4968: Chemotherapy up-regulates RANK-RANKL signaling in breast cancer cells.
Authors: Sharma Bhawna , Varney Michelle L. , Singh Rakesh K.
Published: August 2014

Chemotherapy has been the mainstay of cancer treatment for so many years and so do the increasing resistance to it. Recent studies emphasize the importance of cytokines in breast cancer therapy resistance and how targeting cytokines or their receptor may prove to be a better therapeutic regimen. This study focuses on the role of receptor activator of NFkB ligand (RANKL), recently been observed to provide survival benefits to cancer cells and helps in mammary stem cell maintenance. To the best of our knowledge the modulations of RANK and RANKL in response to chemotherapy has not been evaluated before. Our preliminary studies with drug-resistant cells showed higher expression of RANK and RANKL both in vitro in cells as well as in vivo in tumors formed by these cells, suggesting a role of this RANKL mediated signaling in breast cancer therapy resistance. Given that RANK and RANKL are expressed in aggressive breast tumors and this signaling provides survival benefits to cancer cells, we hypothesized that chemotherapy up-regulates RANK-RANKL signaling in breast cancer cells which eventually makes breast cancer cells resistant to further chemotherapy providing evasion to therapy. To investigate this, we monitored the expression of RANKL in murine 4T1, CL66 and MDA MB-231, MCF-7; human breast cancer cells lines after treatment with two commonly used drugs doxorubicin and paclitaxel for breast cancer treatment. We observed that both drugs up-regulate RANKL expression in these cells. To examine if the expression of RANK also changes after drug treatment in these cells, we treated 4T1 and Cl66 cells with paclitaxel for 4, 8 and 12 hours. We observed an increase in the expression of RANK in these cells in a dose- and duration-dependent manner. 4T1 and Cl66 along with MDA MB-231 and MCF-7 were also treated with both paclitaxel and doxorubicin to evaluate if the change in receptor and ligand expression was drug-dependent. Our data showed that both the drugs up-regulate RANKL at mRNA (qRT-PCR) and protein levels (Immunofluorescence) in Cl66 and 4T1 cells. This up-regulation was as early as 4hrs (at mRNA level) after drugs treatment and the expression decreases after 8 hrs, suggesting an early transcriptional up-regulation. Our data suggest that increased expression of both RANK and RANKL in the breast cancer cells following chemotherapy may contribute to the activation of RANK-RANKL signaling in breast cancer and targeting this signaling might provide a novel adjuvant therapeutic regimen.

Citation Format: Bhawna Sharma, Michelle L. Varney, Rakesh K. Singh. Chemotherapy up-regulates RANK-RANKL signaling in breast cancer 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 4968. doi:10.1158/1538-7445.AM2013-4968
Abstract 4974: Oligoclonal characterization of tumor-infiltrating plasma cells in breast cancer: Germ cell expansion and immunity .
Authors: Shukla Anurag , Sun Yu-Jing , Krag David
Published: August 2014

Breast cancer is the most common invasive cancer in women in the United States and many other parts of the world. About 80% of all the breast cancers are Invasive Ductal Carcinomas (IDC). Breast cancers often show detectable lymphocytic infiltrates, and only about one-fourth of them contain moderate to heavy infiltration of B-cells and plasma cells. B cells are the effector cells of humoral immunity and can terminally differentiate into antibody secreting plasma cells. In the present study, we have analyzed the immunoglobulin variable genes of tumor-infiltrating plasma cells from IDC patients. This was done to determine the clonal character of the plasma cells and their possible role in the tumor immunity. Fresh malignant breast tumors were obtained following surgery, and their frozen sections were screened for the lymphocytic infiltration. The tumor samples from patients which showed good plasma cell-infiltration were used for this study. Both malignant breast tumors, A55 (estrogen receptor-positive 80%; progesterone receptor-positive >90%) and A91 (estrogen receptor positive 70%; progesterone receptor positive 90%), were categorized as invasive ductal carcinomas. Using a laser capture microdissection technique (PALM Microlaser Technologies AG, Bernried, Germany), single and small clusters of plasma cells in the tumors were collected. The tumor plasma cells were processed for human immunoglobulin variable gene analysis using appropriate RT-PCR primer sets for heavy and light chains. A set of B- cells from healthy volunteer blood was also run as a RT-PCR quality control. The analyses of variable regions (VHeavy and VLight) of immunoglobulin genes show a very limited number of plasma cell clones in both the tumors. These results indicate their origin from a possible B-cell germ center within the tumor. These plasma cells may be secreting antibodies relevant to the tumor antigens. In order to confirm this, we plan to clone these VH and VL genes and produce antibodies in vitro and examine their binding to the tumor from which they were derived. The work will be extended to study the effects of these antibodies on breast cancer cell proliferation.

Citation Format: Anurag Shukla, Yu-Jing Sun, David Krag. Oligoclonal characterization of tumor-infiltrating plasma cells in breast cancer: Germ cell expansion and immunity . [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 4974. doi:10.1158/1538-7445.AM2013-4974
Abstract 4980: Myeloid cells display immunosuppression activities during pregnancy, participating to the establishment of pre-metastatic niches.
Authors: Le Bitoux Marie-Aude , Waeber Sabine , Stamenkovic Ivan
Published: August 2014

During pregnancy, major physiological changes occur that include inflammation and reduced immunity, conditions that are both implicated in promoting tumor growth. Tumors are indeed more aggressive during pregnancy and have greater metastatic proclivity. To gain insight into the mechanisms that may render pregnancy permissive for tumor growth, we addressed the biological and functional properties of myeloid-derived suppressor cells (MDSC), a heterogeneous population of hematopoietic cells of variable plasticity and immunoregulatory properties, during mouse and human gestation.

Gene expression profiling of mouse MDSC has been shown to differ in pregnant and virgin mice, and the gene expression profile of pregnant animal-derived MDSC bears similarity to that observed in MDSC of tumor bearing mice. Common induced genes include Fibronectin1 and Olfactomedin4, which are known to be involved in extracellular matrix remodeling and tissue permissiveness to tumor cell implantation.

In human pregnancy, CD33+ myeloid cells isolated from peripheral blood mononuclear cells (PBMC) are shown to be more strongly inhibitory of T cell proliferation in pregnant than in non-pregnant women. We also observed reduced natural killer cell numbers in the circulation of pregnant women, but which was not linked to a decrease in their cytotoxic activity, even after co-culture with CD33+ PBMC.

Our observations suggest that mouse MDSC, as well as human CD33+ cell populations that arise during the physiological state of gestation, mimic tumor progression-associated MDSC and participate in the constitution of a permissive soil, often referred to as the pre-metastatic niche.

Citation Format: Marie-Aude Le Bitoux, Sabine Waeber, Ivan Stamenkovic. Myeloid cells display immunosuppression activities during pregnancy, participating to the establishment of pre-metastatic niches. [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 4980. doi:10.1158/1538-7445.AM2013-4980
Abstract 4987: Immunoscore of CD8/CD45RO in pre-operative biopsy specimens predicts TNM-stage in primary colorectal cancer.
Authors: Koelzer Viktor H. , Lugli Alessandro , Dawson Heather , Borner Markus , Inderbitzin Daniel , Zlobec Inti
Published: August 2014

Background:

Colonoscopy and biopsy are the standard procedure for primary diagnosis of colorectal cancer (CRC), but prognostic biomarkers based on biopsy material are still missing. The tumor microenvironment has been shown to play a major role in the pathogenesis of CRC. Therefore, we hypothesized that an immunoscore of CD8+ T-effector and CD45RO+ memory T-cell infiltrates in pre-operative biopsy specimens could have an impact on the pre-operative management of CRC patients by predicting TNM-stage.

Methods:

Intraepithelial (i) and stromal (s)CD8+ and CD45RO+ T-cells were visualized in biopsy specimens of 134 well characterized CRC patients by immunohistochemistry and quantified in 5 high-power-fields each of the highest density of immune cell infiltration. Cut-off scores for prediction of pN, pM and pT in the matched resection specimen were established using Classification and Regression Tree (CART) analysis. Infiltration of CD8+ and CD45RO+ T-cells in each compartment was assessed for prediction of TNM stage, lymphovascular invasion and tumor grade.

Results:

High numbers of infiltrating sCD45RO T-cells in biopsy material were associated with earlier T-stage in the matched resection specimen(p<0.0001) as well as with earlier tumor grade (p=0.0223) and absence of distant metastasis (p=0.0177; specificity=62.5% and positive predictive value (PPV)=93%). High total (t) numbers of infiltrating CD8-T cells in biopsy material were strongly predictive of absence of nodal metastasis (p=0.0182) as well as absence of lymphatic invasion (p=0.0201). Combined analysis of tCD8 and tCD45RO with pN led to 100% specificity and PPV. No association of CD8+ and CD45R0+ T-cell-infiltration with vascular invasion was observed. In contrast, absence of both CD8+ and CD45RO+ T-cells in pre-operative biopsies predicted frequent nodal and distant metastatic spread, advanced T-stage and lymphovascular invasion in the matched resection specimen.

Conclusion:

Total CD8 counts and sCD45RO in pre-operative biopsy specimens allows prediction of essential clinical characteristics of primary CRC in the matched resection specimen including full TNM-stage and lymphatic invasion. Analysis of total CD8+ and sCD45RO+ in pre-operative biopsies may thereby provide a decisive indicator for risk stratification of CRC patients in the preoperative setting.

Citation Format: Viktor H. Koelzer, Alessandro Lugli, Heather Dawson, Markus Borner, Daniel Inderbitzin, Inti Zlobec. Immunoscore of CD8/CD45RO in pre-operative biopsy specimens predicts TNM-stage in primary colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2013-4987
Abstract 4990: Complement promotes growth of ovarian cancer.
Authors: Cho Min Soon , Vasquez Hernan G. , Han Hee Dong , Pradeep Sunila , Rupaimoole Rajesh , Zand Behrouz , Kroll Michael , Sood Anil K. , Afshar-Kharghan Vahid
Published: August 2014

Objective: The complement system is a component of innate immunity against invading pathogens, but also mediates the cell-cell communication in organ regeneration, angiogenesis, epithelial-mesenchymal transition (EMT) and cell migration. We studied whether the complement system affect tumor growth in ovarian cancer.

Materials & Methods: Complement C3 mRNA level was quantified using qRT-PCR in ovarian cancer cell lines and in tumor specimens from patients with ovarian cancer. Small interfering RNA (siRNA) was used to investigate the consequences of C3 gene silencing in vitro and in vivo.

Results: C3 was expressed in ovarian cancer cell lines and tumor tissues from patients. Silencing of the C3 gene decreased ovarian cancer cell proliferation, migration and invasion in vitro. In orthotopic murine models of ovarian cancer, C3 siRNA reduced growth of implanted tumors by 60% as was evident by lower number and smaller size of tumor nodules in C3-siRNA treated mice in comparison to those in scrambled siRNA treated control mice (average tumor weight of 0.19 g in C3 siRNA vs. 0.61 g in scrambled siRNA, n=8 in each group, p=0.017; average nodule number of 4.4 in C3 siRNA vs. 11.4 in scrambled siRNA, n=8 in each group, p=0.05; t-test). Consistent with our in vitro and animal studies, ovarian cancer patients with a higher C3 mRNA level in their tumors had a worse prognosis with overall survival rates compared to those with a lower C3 mRNA level (n=75, p=0.03).

Conclusion: Our studies revealed a novel role for complement in cancer biology. Cancer cell-derived C3 promotes tumor growth and progression. Our studies might identify the complement system as a new target for the development of new anti-tumor reagents.

Citation Format: Min Soon Cho, Hernan G. Vasquez, Hee Dong Han, Sunila Pradeep, Rajesh Rupaimoole, Behrouz Zand, Michael Kroll, Anil K. Sood, Vahid Afshar-Kharghan. Complement promotes growth of 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 4990. doi:10.1158/1538-7445.AM2013-4990