bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1348: CD4 T cells targeting citrullinated vimentin reject advanced tumors
Authors: Durrant Lindy G. , Brentville Victoria A. , Metheringham Rachael L. , Daniels Ian , Symonds Peter , Gijon Mohamed , Xue Wei
Published: August 2015

CD4 cells are potent effectors but CD4 responses to tumour associated antigens are attenuated. Cellular stress induces autophagy which leads to modification of proteins recognised by the immune system. In the absence of inflammation, immunity is regulated, whereas in its presence CD4 responses to modified self-antigens are stimulated. T cells commonly target modified self-antigens and have been shown to play a role in the pathophysiology of several autoimmune diseases. In this study the ability of these CD4 cells to target cancer has been explored. One modification is conversion of arginine to citrulline. Peptidylarginine deiminases (PADs), are a family of calcium dependent enzymes found in a variety of tissues, where their role is post translation citrullination of proteins. Citrullination is conversion of the positively charge aldimine group ( = NH) group of arginine to the neutrally charged ketone group ( = O) of citrulline. Immunisation with citrullinated vimentin peptides induced IFNγ and granzyme B secreting CD4 T cells in response to autophagic tumour targets. The induced CD4 responses to citrullinated self-peptide epitopes show minimal reactivity to the unmodified sequence. A single immunization with modified peptide, up to 14 days after tumour implant, resulted in long term survival in 60-90% of animals with no associated toxicity. The anti-tumour responses were dependent upon CD4 but not on CD8 T cells. This study provides the first evidence that tumours can present citrullinated peptides that can be targets for CD4 cells. This presentation is dependent upon autophagy and PAD enzymes. The CD4 T cells release IFNγ and show direct cytotoxicity which results in potent anti-tumour responses in vivo. This approach is being fast tracked into the clinic in patient's whose tumours express vimentin. This could be patients with mesenchymal tumours or in patients whose tumours undergo epithelial to mesenchymal transition as vimentin is one of the first proteins to be expressed in this transition.

Citation Format: Lindy G. Durrant, Victoria A. Brentville, Rachael L. Metheringham, Ian Daniels, Peter Symonds, Mohamed Gijon, Wei Xue. CD4 T cells targeting citrullinated vimentin reject advanced tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1348. doi:10.1158/1538-7445.AM2015-1348
Abstract 1355: Functional inactivation of LKB1 increases the production of pro-inflammatory cytokines and governs response to immune modulation
Authors: Akbay Esra A. , Koyama Shohei , Li Yvonne , Herter-Sprie Grit S. , Thai Tran C. , Aref Amir R. , Soucheray Margaret , Shimamura Takeshi , Barbie David A. , Dranoff Glenn , Hammerman Peter S. , Wong Kwok-Kin
Published: August 2015

LKB1/STK11 is one of the most commonly mutated tumor suppressors in non-small cell lung cancers. Inactivation of LKB1/STK11 is common in lung cancers with mutated KRAS alleles (1) and results in a clinically more aggressive and metastatic phenotype (2-5). Here, we show in genetically engineered mouse models (GEMMs) and in primary human cancer specimens that loss of LKB1/STK11 in the context of mutated KRAS has a marked effect on the tumor immune microenvironment characterized by accumulation of neutrophils and of tumor promoting cytokines including interleukin (IL)-6. Importantly, Kras/Lkb1 tumors also carry reduced number of tumor-infiltrating lymphocytes with the infiltrating lymphocytes expressing higher levels of T cell exhaustion markers and greater proportions of regulatory T cells as compared to KRAS-driven (Kras) tumors. We also found that presence of LKB1 mutation was negatively correlated with programmed death-1 ligand-1 (PD-L1) expression in mouse and human lung tumors and that antibodies therapeutically targeting this immune checkpoint were ineffective in Kras/Lkb1 mice. In contrast, an IL-6 neutralizing antibody showed clinical benefit in the Kras/Lkb1 model via the reduction of neutrophil accumulation and proinflammatory cytokines. These findings suggest that LKB1 loss of function exacerbates the tumor inflammatory microenvironment in lung cancers and that targeting the aberrant cytokine signaling in LKB1-deficient tumors could be a viable therapeutic option.

Citation Format: Esra A. Akbay, Shohei Koyama, Yvonne Li, Grit S. Herter-Sprie, Tran C. Thai, Amir R. Aref, Margaret Soucheray, Takeshi Shimamura, David A. Barbie, Glenn Dranoff, Peter S. Hammerman, Kwok-Kin Wong. Functional inactivation of LKB1 increases the production of pro-inflammatory cytokines and governs response to immune modulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1355. doi:10.1158/1538-7445.AM2015-1355
Abstract 1359: Co-expression of ULBP1 and NKG2D are related to better overall survival in patients with gastric cancer
Authors: Yoshimura Kiyoshi , Inoue Moeko , Kamei Ryoji , Kitano Shigehisa
Published: August 2015

Objective

The relationship between expressions of ULBP1/NKG2D and their clinicopathological outcome in patients with gastric cancer were investigated.

Background

ULBP1 which is one of NKG2D ligand and expressing on tumor cell surface binds to the NKG2D receptor presenting on NK, CD8+ T and gamma delta T cell. It was considered that innate immunity is mainly induced through the interaction between ULBP1 and NKG2D. The role of this immune response is not clear yet, and is controversial whether it is inhibited or activated signal.

Methods

We performed immunohistochemical staining of gastric cancer tissue with antibody against ULBP1 and NKG2D in 98 patients who received surgery in 2004 to 2008. We investigated expression of ULBP1 in tumor and NKG2D in mononuclear cells around tumor by multiple points observation. These results were compared to their association with histological and clinical outcome.

Results

The positive group of NKG2D expression on mononuclear cells around gastric cancer tissue had significantly longer overall survival (OS) than the negative group (p = 0.0217). Although it was not recognized significant difference of ULBP1 expression on gastric cancer cells, in a combination of ULBP1 and NKG2D expression, both positive group had best OS, and both negative group had poorest OS in this study.

Conclusion

The interaction between ULBP1 and NKG2D in gastric cancer may have positive signal which relates to good OS. Although the mechanism of immune system through ULBP1/NKG2D is controversial, we obtained an interesting result according to this mechanism.

Citation Format: Kiyoshi Yoshimura, Moeko Inoue, Ryoji Kamei, Shigehisa Kitano. Co-expression of ULBP1 and NKG2D are related to better overall survival in patients with gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1359. doi:10.1158/1538-7445.AM2015-1359
Abstract 1363: VEGF reduction facilitates angiogenesis by CXCR2
Authors: Kosir Mary A. , Ju Donghong
Published: August 2015

PURPOSE: With multiple pathways for angiogeneis, the purpose of the study is to quantify the expression of angiogenic tyrosine kinase receptors during endothelial tubule formation while inhibiting a G-protein linked CXCR2 receptor that binds angiogenic CXC chemokines.

METHODS: Using endothelial tubule formation assays on Matrigel and Growth Factor Reduced (GFR) Matrigel, and without added VEGF, human umbilical vascular endothelial cells (HUVEC) were treated with a selective small molecule non-peptide inhibitor of CXCR2 called SB225002 (N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl)urea compared with control. Previous dose-inhibition curves established optimal dosing of SB225002. After 6 hours of incubation, endothelial tubule formation was quantified by counting tubule length in four random microscopic fields using the microscope camera software NIS-Elements AR 3.10 in triplicate and compared to control. In parallel, endothelial cells were collected from the Matrigel under the same conditions for quantification of angiogenesis receptors and growth factors by RT-PCR in triplicate.

RESULTS: There was significant inhibition of endothelial tubule formation on Matrigel (and GFR Matrigel) by the addition of SB225002 (40-58% of inhibition) under conditions of no added VEGF when compared to control. The expression of mRNA in HUVEC cells harvested from Matrigel was not different for VEGFR1-3, FGFR1-3, and PDGFRA,B with or without the inhibitor. Under conditions of no added VEGF, the expression of CXCR2 mRNA was the same with or without the inhibitor, but the endothelial tubule formation was significantly inhibited.

CONCLUSION: Under growth factor reduced conditions, including no added VEGF, the endothelial tubule formation and angiogenenic tyrosine kinase receptor expression was similar to standard matrigel. However, when a selective non-peptide inhibitor of CXCR2 (SB225002) was added, endothelial tubule formation was inhibited without change in expression of other angiogenesis receptors from HUVEC cells retrieved from the matrigel. Therefore, tubule formation continues with the chemokine/CXCR2 axis and may be an additional pathway that circumvents other attempts at angiogenesis inhibition targeting tyrosine kinase inhibitors.

Citation Format: Mary A. Kosir, Donghong Ju. VEGF reduction facilitates angiogenesis by CXCR2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1363. doi:10.1158/1538-7445.AM2015-1363
Abstract 1369: Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme
Authors: Staberg Mikkel , Michaelsen Signe Regner , Olsen Louise Stobbe , Nedergaard Mette Kjølhede , Villingshøj Mette , Poulsen Hans Skovgaard
Published: August 2015

The high-grade glioma, Glioblastoma Multiforme (GBM), is the most prevalent and aggressive brain tumour in adults with a median survival of fifteen months. Thus new therapeutic targets are urgently needed. GBM is characterized by extensive vascularisation why targeting angiogenesis has promising potential in improving the treatment of GBM patients. Aberrant EGFR signaling is another characteristic of GBM, and increased EGFR downstream signaling is believed to correlate with poor prognosis. However, little is known of the involvement of EGFR signaling in GBM angiogenesis. Notch signaling is known for its role in angiogenesis during foetal development and in other cancers, but the importance of Notch signaling in GBM angiogenesis is yet to be elucidated. Recently, it has been shown, that crosstalk between Notch- and EGFR signaling occurs in cancer cells, and aberrant EGFR signaling may therefore play a functional role in GBM angiogenesis through crosstalk with Notch signaling.

The aim of this study was to investigate the effect of combined Notch- and EGFR inhibition on GBM cell viability and on ability of GBM cells to induce angiogenesis in endothelial cells.

In vitro assays of GBM angiogenesis were established using two patient derived GBM cell cultures, one expressing the mutated EGFR, the EGFRvIII, and one expressing wild type EGFR. The functional relevance of EGFR- and Notch signaling in GBM cells was tested using the tyrosine kinase inhibitor Iressa for EGFR inhibition, and the gamma-secretase inhibitor DAPT for Notch inhibition. We show that combined inhibition of EGFR- and Notch signaling caused additive inhibition of cell viability of both cell cultures in vitro. Abrogated EGFR/Notch signaling in GBM cells further reduced angiogenesis in endothelial cells exposed to conditioned media from treated GBM cells. Moreover, when GBM cells were treated with the inhibitors, this inhibited phosphorylation and activation of the two survival signalling pathways ERK and Akt, and abrogated secretion and expression of the most prominent angiogenic factor - VEGF. The presented results indicate that EGFR- and Notch signaling play a functional role in GBM angiogenesis and that combined treatment with drugs targeting both the EGFR- and Notch pathway could prove beneficial over single drug therapy in targeting both the tumorigenic and angiogenic potential of GBM cells.

Citation Format: Mikkel Staberg, Signe Regner Michaelsen, Louise Stobbe Olsen, Mette Kjølhede Nedergaard, Mette Villingshøj, Hans Skovgaard Poulsen. Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1369. doi:10.1158/1538-7445.AM2015-1369
Abstract 138: Novel PAK4 allosteric modulators (PAMs) provide potential therapeutic options in human gastric cancer
Authors: Chien Wenwen , Xiao Jinfen , Ding Ling-Wen , Muzakar Muhammad Ikhsan B. , Sun Qiao-Yang , Gery Sigal , Senapedis William , Shacham Sharon , Baloglu Erkan , Koeffler H. Phillip
Published: August 2015

Expression of PAK4 (p21-activated serine/threonine kinase 4) is associated with gastric tumorigenesis. Overexpression of PAK4 is correlated with metastasis of gastric cancer and in part confers cisplatin resistance in gastric cancer patients. We transduced the human gastric cancer cell line AGS with CRISPR/cas9 targeting PAK4, and observed reduced cell proliferation and motility. Novel PAK4 allosteric modulators (PAMs) were evaluated for their potential therapeutic use for gastric cancer. A panel of human gastric cancer cell lines was screened with four PAMs (KPT-7189, -7349, -7657, -8752). MTS assay showed inhibition of cell viability by PAMs in 6 of 10 gastric cancer cell lines (IC50 ranged from 50 to 5000 nM). Cell cycle analysis and annexin V assay indicated a combination of cell growth arrest at G1 phase and cell apoptosis after treatment with PAMs. Furthermore, PAMs diminished expression levels of PAK4, induced apoptosis-related molecule caspase-3, and suppressed PI3K/Akt and MEK/Erk signaling. Wound assay showed inhibition of cell migration and soft agar assay demonstrated suppression of colony formation for gastric cancer cells treated with PAMs. Mouse xenograft models are currently under investigation. Remarkably, PAMs in combination with cisplatin induced cell death in cisplatin-resistant gastric cancer cells in a synergistic manner. This study demonstrates PAMs have anti-tumor activity against gastric cancer cells and suggests their potential use for the treatment of gastric cancer.

Citation Format: Wenwen Chien, Jinfen Xiao, Ling-Wen Ding, Muhammad Ikhsan B. Muzakar, Qiao-Yang Sun, Sigal Gery, William Senapedis, Sharon Shacham, Erkan Baloglu, H. Phillip Koeffler. Novel PAK4 allosteric modulators (PAMs) provide potential therapeutic options in human gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 138. doi:10.1158/1538-7445.AM2015-138
Abstract 1386: KLF5 inhibits angiogenesis in PTEN-deficient prostate cancer by attenuating AKT activation and subsequent HIF1α accumulation
Authors: Ci Xinpei , Xing Changsheng , Zhang Baotong , Zhang Zhiqian , Ni Jenny Jianping , Zhou Wei , Dong Jin-Tang
Published: August 2015

Background: KLF5 is a basic transcriptional factor that regulates multiple physiopathological processes. Our recent study showed that deletion of Klf5 in mouse prostates promotes tumorigenesis initiated by the deletion of Pten. While molecular characterization of Klf5-null tumors suggested that angiogenesis was partially responsible for tumor promotion, the precise function and mechanism of KLF5 deletion in prostate tumor angiogenesis remain unclear.

Results: Using histological staining of Pten-null mouse prostates, we observed that deletion of Klf5 significantly increased the number of microvessels, which was accompanied by the upregulation of multiple angiogenesis-related genes based on microarray analysis with MetaCore software. In human umbilical vein endothelial cells (HuVECs), tube formation and migration, both of which are indicators of angiogenic activities, were decreased by conditioned media from PC-3 and DU 145 human prostate cancer cells with KLF5 overexpression, but increased by media from cells with KLF5 knockdown. HIF1α, a key angiogenesis inducer, was upregulated by KLF5 loss at the protein but not the mRNA level in both mouse tissues and human cell lines, as examined by immunohistochemical staining, real-time RT-PCR and Western blotting. Consistently, KLF5 loss also upregulated VEGF and PDGF, two pro-angiogenic mediators of HIF1α function, as analyzed by immunohistochemical staining in mouse tissues and ELISA in conditioned media. Mechanistically, AKT activity, which caused the accumulation of HIF1α, was increased by KLF5 knockout or knockdown but decreased by KLF5 overexpression. Consistently, PI3K/AKT inhibitors abolished the effects of KLF5 knockdown on angiogenic activity, HIF1α accumulation, and VEGF and PDGF expression.

Conclusion: KLF5 loss enhances tumor angiogenesis by attenuating PI3K/AKT signaling and subsequent accumulation of HIF1α in PTEN deficient prostate tumors.

Citation Format: Xinpei Ci, Changsheng Xing, Baotong Zhang, Zhiqian Zhang, Jenny Jianping Ni, Wei Zhou, Jin-Tang Dong. KLF5 inhibits angiogenesis in PTEN-deficient prostate cancer by attenuating AKT activation and subsequent HIF1α accumulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1386. doi:10.1158/1538-7445.AM2015-1386
Abstract 1400: Evaluation of tumor-initiating cell populations in the KPC mouse model of pancreatic cancer
Authors: Dosch Joseph S. , Ziemke Elizabeth K. , Shettigar Amrith , Rehemtulla Alnawaz , Sebolt-Leopold Judith S.
Published: August 2015

There continue to be many questions about the frequency and phenotype of tumor-initiating cells or cancer stem cells (CSCs) which may play an important role in cancer metastasis and drug resistance in several tumor types, including pancreatic cancer. The KrasG12D, Trp53R172H, and Pdx-1-Cre (KPC) induced model of pancreatic cancer has gained considerable attention for its ability to replicate the clinical features of the disease. Here, we report the generation of a novel syngeneic xenograft model of luciferase-tagged pancreatic tumor cells (KPCL) to facilitate the investigation of the tumorigenic capacity of this cancer model. Limiting dilution analysis of bulk KPCL tumor cells revealed that 1/87 cells were capable of forming new tumors, a number considerably higher than reported for human pancreatic adenocarcinomas. Cell surface markers CD44, CD24, CD133, c-Met, and functional ALDEFLUOR activity, previously shown to mark CSC populations in human and mouse pancreatic tumors, were heterogeneous across a large panel of spontaneous KPCL tumors. Additional testing of CD133 and ALDEFLUOR populations were unable to enrich for tumorigenic populations when transplanted into syngeneic mice. Additionally, spheroid cultures initiated from KPCL tumors did not enrich for cells with stem-like characteristics and were not significantly more tumorigenic than KPCL cells cultured as monolayers. These studies demonstrate that the tumor-forming potential of KPC cells is common, and may not fit the hierarchically organized CSC model that is reported for the human disease.

Citation Format: Joseph S. Dosch, Elizabeth K. Ziemke, Amrith Shettigar, Alnawaz Rehemtulla, Judith S. Sebolt-Leopold. Evaluation of tumor-initiating cell populations in the KPC mouse model of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1400. doi:10.1158/1538-7445.AM2015-1400
Abstract 1408: Generation of 3D-microtissues suitable for drug screening with lentivirally GFP-labelled CD44+CD24- breast cancer cells enriched by irradiation
Authors: Höfig Ines , Rosemann Michael , Albrecht Michaela , Ingawale Yashodhara , Kelm Jens M. , Atkinson Michael J. , Thirion Christian , Anastasov Natasa
Published: August 2015

A stem cell like subpopulation of human T47D and MDA-MB-361 breast cancer cells (CD44+CD24-) was enriched by irradiation (2 Gy) and isolated using magnetic activated cell sorting. As an additional stem cell model, primary murine mesenchymal stem cells (MSCs) were isolated from the bone marrow of C3HxBL mice. We confirmed the stemness phenotype by repopulating heterogeneous cancer cell lines from isolated CD44+CD24- cells or by inducing differentiation of MSCs to the osteocyte, adipocyte and chondrocyte lineage.

For gene delivery in cancer research, lentiviral transduction protocols are widely used though low transduction efficiencies of especially primary cells force the use of high lentiviral titers. In search for an optimized transduction protocol we came across a poloxamer-based chemical adjuvant to increase lentiviral gene delivery in primary cells [Höfig et al, Journal of Gene Medicine, 2012 and Höfig et al, Biomaterials, 2014]. Next we aimed to expand these benefits to transduce cells with a stem cell phenotype. After lentiviral transduction of a GFP encoding lentivirus in the presence of the poloxamer-based chemical adjuvant we could detect highest numbers of GFP expressing cells. Neither the chemical adjuvant nor the transgenic GFP expression altered the stemness phenotype of those cells analysed by repopulation or differentiation assays and by detection of stem cell markers on mRNA level (CD133 and Sox2). To monitor the fate of GFP-expressing CD44+CD24- cells, we cultured them in 3D-microtissues in mix with unlabelled bulk cells and analysed the effect of irradiation on cell number and location of CD44+CD24- cells within the spheroid.

In this study we present a method for induction and isolation of human breast cancer cells with a stem cell like phenotype and developed an improved lentiviral transduction method by a chemical adjuvant preserving the stemness character in human and murine cells. Efficient lentiviral transduction enabled the generation of 3D-microtissues with GFP-labeled CD44+CD24- cells that both can be studied in cancer research and shall have applications for drug screening.

Citation Format: Ines Höfig, Michael Rosemann, Michaela Albrecht, Yashodhara Ingawale, Jens M. Kelm, Michael J. Atkinson, Christian Thirion, Natasa Anastasov. Generation of 3D-microtissues suitable for drug screening with lentivirally GFP-labelled CD44+CD24- breast cancer cells enriched by irradiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1408. doi:10.1158/1538-7445.AM2015-1408
Abstract 1436: The SxxSS motif of T-cell Factor-4 isoforms regulates Wnt5a expression and EMT in human liver cancer cells
Authors: Koga Hironori , Nakamura Toru , Abe Mitsuhiko , Ikezono Yu , Wada Fumitaka , Yano Hirohisa , Torimura Takuji
Published: August 2015

Background: The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. Previously we identified 14 TCF-4 isoforms derived from human HCC cell lines (Exp Cell Res 2011). The TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of a SxxSS motif. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012). However, K cells often showed morphological alteration, reminiscent of epithelial-mesenchymal transition (EMT), which is involved in non-canonical Wnt signaling (BMC Cancer 2013). The finding suggested that the SxxSS motif had potential to regulate EMT through the non-canonical Wnt signaling pathway. Thus, the AIM of this study was to investigate whether the SxxSS motif modulated expression levels of EMT regulators and Wnt5a, a representative non-canonical Wnt ligand. Methods: The human HCC cell line HAK-1A (Hepatology 1993) was used. TCF-4K mutants (269A, 272A, and 273A) were prepared with conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (269A, 272A, and 273A cells, respectively) were established. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Sh-RNA was used to knockdown wnt5a gene expression. Results: The 269A-mutant cells robustly expressed Wnt5a in both protein and mRNA levels, while empty vector-transfected cells (control), J cells, or K cells did not. Of note, Wnt5a expression was coupled with SLUG expression and EMT-like cellular morphological change. Snail was hardly expressed in the cells examined in this study. When the Wnt5a expression was specifically silenced by using sh-RNA, the expression level of SLUG was clearly decreased. Conclusion: The findings in this study suggest that serine 269 (the first serine) of the SxxSS motif of TCF-4 is a major switch to control Wnt5a transcription, thereby modulating the expression level of the EMT-regulator SLUG in a human HCC cell line.

Citation Format: Hironori Koga, Toru Nakamura, Mitsuhiko Abe, Yu Ikezono, Fumitaka Wada, Hirohisa Yano, Takuji Torimura. The SxxSS motif of T-cell Factor-4 isoforms regulates Wnt5a expression and EMT in human liver cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1436. doi:10.1158/1538-7445.AM2015-1436
Abstract 1442: Understanding the role of autophagy during cancer metastasis
Authors: Lim Maegan M. , Teo Ziqiang , Choo Chee Chong , Zhu Pengcheng , Tan Nguan Soon
Published: August 2015

Introduction:

Autophagy is a catabolic process that involves the lysosomal degradation and recycling of cellular components. It acts as a cell protective mechanism to promote cellular homeostasis or survival during nutrient deprivation. Studies in the past decade have shown the involvement of autophagy during tumorigenesis, but much are left to be debated on its controversial role as a tumour suppressor or a potential protective role in promoting cancer cell survival. Knockout of autophagic core machinery proteins in mice models showed increase potential of tumorigenic development. Recent findings also suggested that autophagy plays a part during cancer cell metastasis, but till date, there is no significant evidence to clearly define the involvement of autophagy and the mechanistic behind it. Our study involves defining the role of autophagy during metastasis and the molecular drivers behind the process.

Material & Methods:

Using human squamous cell carcinoma cell lines, we induce epithelial-mesenchymal transition (EMT) with three different methods to study the change in the autophagy level during the process.

Results:

Our unpublished data have demonstrated a higher autophagy level during our three models of EMT induction. Guided by the microarray data, we investigated the molecular drivers behind the change in autophagy levels and showed evidence that knocking down of certain molecular drivers reverses the effect and delay EMT.

Citation Format: Maegan M. Lim, Ziqiang Teo, Chee Chong Choo, Pengcheng Zhu, Nguan Soon Tan. Understanding the role of autophagy during cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1442. doi:10.1158/1538-7445.AM2015-1442
Abstract 1446: Induction of epithelial-to-mesenchymal transition (EMT) by SL-15 knockdown contributes to anoikis resistance in human colorectal cancer metastases
Authors: Jun Soo Young , Cho Hyun-Soo , Lee Jeong-Ju , Ahn Jun-Ho , Yoon Ji-Yong , Lee Jae-Hye , Choi Min Hyuk , Kim Nam-Soon
Published: August 2015

Purpose of the study: Metastasis or systemic dissemination of cancer cells into secondary organs is responsible for 90% of cancer-related fatalities. Our team previously screened out SL-15 as therapeutic target of gastric cancer through the analysis of gene expression profiling. In this study, we investigated the roles of human SL-15 in colorectal cancer (CRC) metastasis. Experimental procedures: SL-15 levels of metastatic liver CRC tissues and of primary CRC tissues as well as of adjacent nontumour tissues were compared using quantitative RT-PCR. Transwell and wound healing methods were used to determine the migration/invasion abilities of CRC cells. Flow cytometery analysis was performed to measure the apoptotic ratio as well as the levels of E-cadherin/p53/SL-15. Confocal observation and Western blot analysis were used for studying the underlying mechanisms of small interfering RNA against SL-15 (siSL-15)-induced CRC metastasis. Results: The level of SL-15 was higher in the order of metastatic liver CRC tissues, primary CRC tissues and then the adjacent nontumor tissues. siSL-15 significantly induced the migration/invasion of CRC cells that was almost entirely suppressed by the co-expression of SL-15. Importantly, the application of siSL-15 contributes to anoikis resistance of CRC cells via the upregulation of zinc finger E-box-binding homeobox 1 (ZEB1) in stem-like subtype CRC cells and intestinal trefoil factor (ITF) in goblet- like subtype CRC cells by the inhibition of glycogen synthase kinase 3 β (GSK3β) and of β-catenin degradation, thereby leading to downregulation of E-cadherin as well as p53 degradation. Importantly, the interactions among SL-15/p53/GSK3β that were dissociated upon the application of siSL-15 were determined. Conclusions: SL-15 is more highly up-regulated in metastatic liver CRC cancer than in primary CRC cancer and the adjacent nontumor tissue: SL-15 knockdown contributes to anoikis resistance of CRC cells and CRC metastasis via inducing epithelial-to-mesenchymal transition as well as p53 degradation due to the dissociation of SL-15 with GSK3β and p53.

Citation Format: Soo Young Jun, Hyun-Soo Cho, Jeong-Ju Lee, Jun-Ho Ahn, Ji-Yong Yoon, Jae-Hye Lee, Min Hyuk Choi, Nam-Soon Kim. Induction of epithelial-to-mesenchymal transition (EMT) by SL-15 knockdown contributes to anoikis resistance in human colorectal cancer metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1446. doi:10.1158/1538-7445.AM2015-1446
Abstract 145: HOTTIP, an oncogenic long non-coding RNA, is frequently up-regulated in hepatocellular carcinoma and is negatively regulated by tumor suppressive microRNA miR-125b
Authors: Tsang Felice H. , Au Sandy L. , Wei Lai , Fan Dorothy N. , Lee Joyce M. , Wong Carmen C. , Ng Irene O. , Wong Chun-Ming
Published: August 2015

Hepatocellular carcinoma (HCC) is one of the most common human cancers. Due to the lack of early diagnostic and effective treatment modalities, the mortality of HCC is high. The development and progression of HCC is a multistep process driven by the accumulation of genetic and epigenetic alterations. Recently, emerging evidence has suggested the role of long non-coding RNAs (lncRNAs) in human carcinogenesis. Herein, by profiling the expression of 88 well-annotated lncRNAs, we identified that HOTTIP was the most significantly up-regulated lncRNA (81%, 57/70) in human HCCs, even in early stage of HCC formation. Functionally, knockdown of HOTTIP attenuated HCC cell proliferation in vitro and markedly abrogated tumorigenicity in vivo. In addition, knockdown of HOTTIP also inhibited migratory ability of HCC cells and significantly abrogated lung metastasis in orthotopic implantation model in nude mice. HOTTIP is an antisense lncRNA mapped to the distal end of the HOXA gene cluster. Knockdown of HOTTIP significantly suppressed the expression of a number of HOXA genes. Furthermore, we identified miR-125b as a post-transcriptional regulator of HOTTIP. Ectopic expression of miR-125b abolished HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Moreover, in human HCCs, HOTTIP expression negatively correlated with that of miR-125b. In Summary, our findings suggest that HOTTIP is a novel oncogenic lncRNA, with miR-125b being its negative regulator. It contributes to hepatocarcinogenesis by regulating the expression of its neighboring protein-coding genes.

Citation Format: Felice H. Tsang, Sandy L. Au, Lai Wei, Dorothy N. Fan, Joyce M. Lee, Carmen C. Wong, Irene O. Ng, Chun-Ming Wong. HOTTIP, an oncogenic long non-coding RNA, is frequently up-regulated in hepatocellular carcinoma and is negatively regulated by tumor suppressive microRNA miR-125b. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 145. doi:10.1158/1538-7445.AM2015-145
Abstract 1466: Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through the incorporation of murine stromal elements
Authors: Delitto Daniel , Pham Kien , Vlada Adrian C. , Sarosi George A. , Thomas Ryan M. , Behrns Kevin E. , Liu Chen , Hughes Steven J. , Wallet Shannon M. , Trevino Jose G.
Published: August 2015

Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma (PC). Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived PC xenografts, establishing the rapidity by which murine cells are integrated into networks of invading cancer cells. In addition, we provide methodology and observations in the establishment and maintenance of a patient-derived PC xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into NOD-SCID mice. Patient demographics, staging, pathologic analysis and outcomes were analyzed. After successful engraftment of tumors, histologic and immunofluorescent analyses were performed on explanted tumors. PC specimens were successfully engrafted in 15 of 25 (60%) of attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages and tumors retain metastatic potential. Interestingly, despite morphologic similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions.

Citation Format: Daniel Delitto, Kien Pham, Adrian C. Vlada, George A. Sarosi, Ryan M. Thomas, Kevin E. Behrns, Chen Liu, Steven J. Hughes, Shannon M. Wallet, Jose G. Trevino. Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through the incorporation of murine stromal elements. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1466. doi:10.1158/1538-7445.AM2015-1466
Abstract 1495: Quantification of blood perfusion in rhabdomyosarcoma xenograft in 3D using contrast-enhanced ultrasound imaging
Authors: Shirinifard Abbas , Thiagarajan Suresh , Sablauer András
Published: August 2015

This study aims to estimate individual 3D perfusion maps using nonlinear contrast-enhanced ultrasound imaging (CEUS) over the volume of individual rhabdomyosarcoma xenograft tumors in CD1 nude mice. We implanted the tumors subcutaneously close to the right kidney. We administered MicroMarker® microbubbles via tail vein catheter and acquired images using VisualSonics VEVO 2100 imaging system. We used the burst-replenishment technique to image tumor perfusion. To maintain a steady concentration of microbubbles, we used a programmable pump to inject a small bolus followed by constant infusion, achieving a steady state in less than 1 min, significantly shorter than the commonly used constant infusion without an initial bolus. Our preliminary analysis showed that the nonlinear CEUS signal intensities of kidney cortex have less than 20% variation between mice. We used a custom program to acquire the CEUS perfusion images over a 3D volume that included the tumor and the right kidney. We used the kidney as a reference organ to normalize whole tumor perfusion data. We applied temporal clustering methods to identify regions with similar perfusion profiles. We fitted three perfusion models to each region: a) mono-exponential, b) lognormal, c) beam-specific. We compared the performance of the models based on their overall quality of fit (across all the regions) and their error in estimated pre-burst signal level. Our preliminary analysis showed that log-normal and multi-vessel beam-specific models provide better performance than the mono-exponential model. We estimated individual 3D perfusion maps based on the model fits. These perfusion maps over the entire tumor volume represent tumor perfusion more accurately than the commonly used methods based on a single 2D plane over a region of interest and without including a reference organ. Our approach quantifies intratumoral blood perfusion heterogeneity and enables comparison of perfusion across individuals. Our method is extensible to other animal models and cancer types provided they can be imaged using ultrasound and a reference tissue can be imaged reliably in tandem on a subset of planes containing the tumor.

Citation Format: Abbas Shirinifard, Suresh Thiagarajan, András Sablauer. Quantification of blood perfusion in rhabdomyosarcoma xenograft in 3D using contrast-enhanced ultrasound imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1495. doi:10.1158/1538-7445.AM2015-1495
Abstract 1496: Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging
Authors: Thiagarajan Suresh , Shirinifard Abbas , Jacus Megan O. , Davis Abigail D. , Patel Yogesh T. , Throm Stacy L. , Daryani Vinay , Stewart Clinton F. , Sablauer András
Published: August 2015

This study quantifies tumor perfusion in individual tumors to estimate blood flow and blood volume parameters of an individualized tumor compartment of a comprehensive physiologically-based pharmacokinetic model of topotecan using an orthotopic xenograft model of pediatric neuroblastoma. We non-invasively imaged perfusion in orthotopic neuroblastoma (NB5) xenograft tumors (n = 3 CD1 nude mice/time point) using nonlinear contrast enhanced ultrasound technique (CEUS). Tumor tissue and organs from the mice were harvested at predefined time-points. We used a programmable syringe pump to inject MicroMarker® microbubbles via tail vein catheter and acquired images using VisualSonics VEVO 2100 imaging system. We used the burst-replenishment technique to image tumor perfusion, which requires a constant concentration of microbubbles in blood during acquisition. To maintain a steady concentration of microbubbles, we programmed the pump to inject a small bolus followed by constant infusion. Our preliminary analysis showed that healthy kidneys rapidly reach a steady state in less than 1 min, significantly shorter than the commonly used constant infusion without an initial bolus. The nonlinear CEUS signal intensities of kidney cortex showed less than 20% variation between mice. We used a custom program to acquire the CEUS perfusion images over a 3D volume that included the tumor and a kidney. We used the kidney as a reference organ to normalize whole tumor perfusion data. We fitted the log-normal perfusion model to estimate perfusion parameters for individual tumors. Our perfusion quantification over the entire tumor volume represents tumor perfusion more accurately than the commonly used methods based on a single 2D plane without a reference organ. Our approach provides population estimates of blood perfusion based on properly normalized estimates of individual blood perfusion parameters.

Citation Format: Suresh Thiagarajan, Abbas Shirinifard, Megan O. Jacus, Abigail D. Davis, Yogesh T. Patel, Stacy L. Throm, Vinay Daryani, Clinton F. Stewart, András Sablauer. Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2015-1496
Abstract 1504: Quantification of dedifferentiation rates in breast cancer using fluorescent reporter constructs and lineage tracing
Authors: Brooks Michael D. , Wicha Max S.
Published: August 2015

Breast cancer is the second most common cancer in women and despite significant advances in treatment, once metastatic it is almost universally fatal. One explanation for this is that current treatments don't target the true driver of tumor growth, cancer stem cells (CSCs). It has been shown that many cancers, including breast cancer, are hierarchically organized with CSCs driving tumor growth. It is also well known that a hallmark of tumor biology is an increased phenotypic plasticity, which helps tumor cells to evade treatment. It has been hypothesized that one consequence of this is an increased rate of dedifferentiation of bulk tumor cells back to CSCs. While a good deal of research has shown that this can sometimes occur, recent work showing the existence of multiple CSC states in breast cancer combined with a lack of direct lineage tracing experiments merits further investigation into the rate, and biological significance, of dedifferentiation in breast cancer. In order to monitor movement between cell states, we have developed a number of fluorescent reporter constructs for genes important in breast cancer including stemness genes (CD44, ALDH1A1, ALDH1A3, Sox2, Oct4), luminal genes (ESR1, Gata3, KRT19, E-Cadherin), myoepithelial/basal genes (CD10, p63, KRT7, Vimentin), and proliferation genes (Ki67, PCNA). These genes will be used in combination with each other in Cre-Recombinase mediated LoxP-STOP-LoxP lineage tracing constructs to track movement between luminal or basal bulk tumor cells and cancer stem cells. Validation of ALDH1A1 and ALDH1A3 reporters by immunofluorescent staining and the AldeFluor Assay has shown good concordance between the reporters and native expression of the genes. These reporters will help to quantify the rate of dedifferentiation and cell state flux in breast cancer.

Citation Format: Michael D. Brooks, Max S. Wicha. Quantification of dedifferentiation rates in breast cancer using fluorescent reporter constructs and lineage tracing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2015-1504
Abstract 1509: Leptin drives mammary stem cell self-renewal via the mTOR pathway
Authors: Esper Ray , Dame Michael , McClintock Shannon , Simon Becky , Wicha Max , Brenner Dean
Published: August 2015

Normal tissue stem cells exist in either a quiescent or proliferating/self-renewing phenotypic state with different patterns of gene expression. In the breast, the CD44+/CD24− cells are retained in a quiescent state, while the ALDH+ stem cells are actively self-renewing cells; the quiescent CD44+/CD24− cells must transition through a self-renewing ALDH+ state in order to differentiate into mature mammary cells. The adipose-rich stroma that makes up the stem cell niche in the human breast is a source of leptin that may alter stem cell phenotype in a paracrine manner. A larger population of proliferating breast stem cells could, in turn, increase the risk of breast cancer. We previously demonstrated that the increased local leptin/adiponectin ratio that commonly occurs in breast tissue of obese women promotes increased breast stem cell self-renewal leading to a larger population of stem cells in vivo, and that the mTOR pathway is activated in normal human mammary stem cells by physiologic concentrations of adipokines, including leptin. There was a physiologic dose response, with a measurable increase in mTOR activity over baseline with 31.25 ng/ml leptin, and a near maximal effect with 125 ng/ml. Here, we isolated the quiescent and proliferating stem cell fractions from human reduction mammoplasty samples and measured basal mTOR activity by ELISA for phosphorylated S6 ribosomal protein (pS6/tS6 ratio), which was greater in the proliferating ALDH+ cells. The quiescent stem cell fraction was treated with physiologic concentrations of leptin prior to resorting with FACS to measure a transition from a CD44+/CD24− to an ALDH+ proliferating phenotype. Parallel quiescent stem cells treated with the mTOR inhibitor everolimus simultaneously with leptin demonstrated that mTOR activity is necessary for this phenotypic switch. If inflammatory adipokines promote stem cell symmetric self-renewal by activating mTOR, targeted interventions may maintain potential tumor-initiating cells in a quiescent state and reduce cancer incidence.

Citation Format: Ray Esper, Michael Dame, Shannon McClintock, Becky Simon, Max Wicha, Dean Brenner. Leptin drives mammary stem cell self-renewal via the mTOR pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1509. doi:10.1158/1538-7445.AM2015-1509