bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1361: Sarcoma-relevant genetic events identified in a chimeric mouse model of sarcoma in skeletal muscle
Authors: Hettmer Simone , Lindsay Melissa C. , Langenau David M. , Wagers Amy J.
Published: August 2012

Background. Soft-tissue sarcomas are heterogeneous malignancies of mesodermal, non-hematopoietic origin. Ex-vivo genetic alteration (by activation of Kras(G12V) and deletion of p16INK4A/p19ARF) and intramuscular implantation of discrete mesodermal cell populations, freshly isolated from mouse skeletal muscle, results in the rapid induction of pleomorphic sarcomas. This model system can be utilized as a platform to dissect sarcoma-relevant cellular and genetic events. Methods. The molecular underpinnings of Kras; p16p19null sarcomas were evaluated by whole genome microarray in comparison to normal muscle and to previously published human rhabdomyosarcoma datasets. Upregulation of 141 candidate genes identified by bioinformatic analyses was verified using a customized RT*2 Profiler™ PCR Array (SA Biosciences). The effects of shRNA knockdown of candidate genes on sarcoma growth were evaluated in sarcoma cell lines. Results. Kras; p1619null sarcomas recapitulate gene expression signatures of human rhabdomyosarcomas and identify a conserved cluster of 144 genes that is concordantly upregulated in both mouse and human sarcomas compared to normal skeletal muscle. 74 of these 144 genes have been associated previously with human malignancies, and 28 have been linked specifically to sarcomas. PCR array validation confirmed that 104 of these genes are upregulated in mouse sarcomas compared to normal skeletal muscle, including the BMP antagonist and Shh morphogen Gremlin, previously implicated in human sarcomas (upregulated 1327-fold in mouse sarcomas vs. muscle). Gremlin knockdown in sarcoma cell lines by shRNA results in reduced proliferation, thereby implicating a functional role in sarcoma biology. Conclusions. Our studies identify a small group of genes that are upregulated in sarcomas across species and may be of fundamental importance in sarcomas. Future studies of the genes identified here will likely provide important insights into the events that drive sarcomas in muscle, and identify new targets for therapeutic intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1361. doi:1538-7445.AM2012-1361
Abstract 1362: Developing genetically flexible mouse models of sarcoma - a novel functional genomics platform
Authors: Gupta Amar , Menezes Serena , Kabaroff Leah , Dickson Brendan , Swallow Carol , Gladdy Rebecca A.
Published: August 2012

Introduction: Sarcomas are a heterogeneous group of malignant neoplasms that arise from connective tissues and have a disease-specific mortality of 30-50%. Existing mouse models of sarcomagenesis utilize knockout and transgenic mice but it is unclear if fixed germline mutations recapitulate sporadic human cancer. In addition, creating germline mutant mice requires substantial time and resources. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS) system would result in sarcomagenesis and could mitigate the limitations of conventional mouse models. Methods: p16/p19 null mice expressing the retroviral receptor, TVA, under nestin (N-tva) or β-actin (BKE) promoters were used. DF1 chicken fibroblasts, infected with avian-leukosis virus vectors (RCAS) carrying combinations of oncogenes (KrasG12D, c-Myc, IGF2) or GFP alone, were delivered by injection into the hindlegs of neonatal mice and tumor development was monitored. Results: GFP-positive cells were observed following in vitro infection of cultured mouse skeletal muscle with RCAS-GFP and GFP+ myoblasts were identified from mice injected with RCAS-GFP in vivo, suggesting successful cell infection and targeted gene delivery. Sarcomas formed in 30% of p16/p19−/−xN-tva mice at the injection site with a median latency of 23.5 weeks (8-42 weeks). Several subtypes of sarcoma developed in this strain based on morphologic assessment and immunohistochemistry (S100, desmin, and smooth muscle actin). Of these, malignant peripheral nerve sheath tumor (MPNST) that overexpressed EGFR consistent with human MPNST, was the predominant subtype (55%). Although a similar tumor incidence occurred in the p16/p19−/−xBKE strain (32%), the median latency was much shorter, 10.4 weeks (7-25 weeks) and the tumor spectrum shifted to predominantly fibrosarcoma (36%) or rhabdomyosarcoma (36%). No tumors were observed in mice injected with empty RCAS vector or without inactivation of the p16/p19 locus. Gene-anchored PCR revealed retroviral vector DNA integration in 89% of N-tva and 97% of BKE tumors. KrasG12D was the most frequent oncogene isolated. Conclusions: We have generated novel models of sarcomagenesis using the RCAS-TVA system to deliver known sarcoma oncogenes into mouse hind legs with a tumor incidence of ∼30%. With optimization, we will be able to use this genetically flexible system to functionally validate novel candidate sarcoma genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1362. doi:1538-7445.AM2012-1362
Abstract 1364: Osteoprotegerin production by breast cancer cells: Regulation by NF-kappaB and role in metastasis
Authors: Weichhaus Michael , Segaran Prabu , Geerts Dirk , Connelly Linda
Published: August 2012

Human breast cancer cells and tissues express the protein osteoprotegerin (OPG). While many studies have investigated the role of OPG in cancer as a mediator of bone remodeling, few studies consider its impact outside the bone microenvironment. Further investigation is therefore required into endogenous OPG expression by breast cancer cells and its effects on tumor progression. OPG can be regulated by the transcription factor NF-kappaB in several cell types. We investigated whether OPG expression in MDA-MB-231 and MDA-MB-435 breast cancer cells was regulated by NF-kappaB. Cells were treated for 24 hours with 10ng/ml TNF-alpha or 10µM thymoquinone (TQ), to stimulate or inhibit NF-kappaB activity respectively. OPG expression was measured by real time RT-PCR (mRNA) and ELISA (protein). OPG levels were increased after treatment with TNF-alpha while treatment with TQ decreased expression. We used the chick embryo metastasis model to determine the effect of OPG on metastasis. Human breast cancer cells were inoculated onto the chick chorioallantoic membrane (CAM) where they form a tumor that metastasizes to the chick tissue. Metastasis is quantified by real time PCR for Human Alu repeat DNA sequences in the chick tissue. We transfected MDA-MB-231 cells with OPG-specific siRNA which reduced OPG mRNA expression up to 72 hours post-transfection. OPG knockdown or control (non-targeting siRNA) treated cells were inoculated onto the CAM at embryonic day 9; chick tissue was harvested at day 16 and analyzed for breast cancer cell metastasis. We found a significant reduction in metastasis of the OPG knockdown cells compared to the control. We performed genome-wide mRNA microarray data analysis to determine whether OPG expression correlates with breast cancer progression in human tumor samples. OPG expression in 1809 breast cancer tissues was not correlated with prognosis. However, analyses by breast cancer subtypes showed that high OPG mRNA expression correlated with poorer prognosis in basal (p=0.0028, n=383) and also HER2 positive tumor subtypes (p=0.021, n=408). In summary, our data suggest that OPG expression in breast cancer cells is regulated by NF-kappaB, a transcription factor associated with pro-tumor effects. Our in vivo studies suggest that OPG promotes metastasis. We are currently determining the mechanism whereby OPG exerts its effects. Since OPG levels correlate with poorer prognosis in basal and HER2 positive tumors, treatment strategies targeting OPG in the primary tumor may represent a potential therapeutic strategy for these disease subytpes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1364. doi:1538-7445.AM2012-1364
Abstract 1366: Development of murine models of breast cancer metastasis for the evaluation of the efficacy of TGF-β inhibitors as therapeutic agents
Authors: Biswas Tanuka , Yang Junhua , Zhao Liu , Sun LuZhe
Published: August 2012

Preclinical models are elemental in understanding drug efficacy and safety in vivo. Many existing models of breast cancer metastasis use immune compromised animals, which are not appropriate for the evaluation of novel therapeutics that modulate host immune response such as inhibitors of transforming growth factor-β (TGF-β) signaling pathway. TGF-β has been shown to be a major immune modulator as well as a major driver in epithelial-mesenchymal transition, which is believed to result in tumor invasion and metastasis. In the current study we characterized a pair of murine mammary cell lines derived from PyMT tumors in C57Bl/6 mice with respect to the effect of TGF-β on their malignant phenotypes. These cell lines, named Py-230 and Py-8119, represent luminal epithelial and mesenchymal cell populations respectively in the tumor. In vitro characterization revealed differences in viability, motility, and transcriptional activation in response to treatment with TGF-β. Py-230 cells were growth inhibited and exhibit epithelial-mesenchymal transition upon treatment with TGF-β whereas Py-8119 was non-responsive to TGF-β in these assays. These observations were consistent with higher Smad activation by TGF-β in Py230 cells than in Py-8119 cells. Furthermore, intra cardiac injections were performed using luciferase-and GFP-transfected PyMT cells in syngeneic animals, which were then treated with either placebo or TβRI KI (kinase inhibitor for TGF-β receptor I). A preliminary study with Py-230 cell-injected mice indicates that TβRI KI treatment appears to enhance bone metastasis. This outcome will be compared with the effect of TβRI KI on the metastatic potential of Py-8119 in our future studies. We believe that the development of mouse models of tumor metastasis using murine mammary cells in a syngeneic background will help illustrate the efficacy of TGF-β inhibitors in abrogating metastasis in an immune competent environment and also allow determining the optimal dose and time of this novel intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1366. doi:1538-7445.AM2012-1366
Abstract 1369: Mouse models of clinical oligo- and poly-metastatic progression
Authors: Zhang Qingbei , Yang Xinan , Shen Jikun , Salama Joseph K. , Khodarev Nikolai , Hasselle Michael D. , Huang Yong , Fan Hanli , Khan Sajid A. , Darga Thomas E. , Hoffma Robert M. , Chmura Steve , Lussier1 Yves A. , Weichselbaum Ralph R. , Weichselbaum Ralph R. , Xing Rosie Hongmei
Published: August 2012

We previously proposed a metastatic state defined by a limited number of metastases, termed oligometastasis(es). These limited metastases may be cured by metastasis-directed treatments in contrast to widespread metastatic disease. While many animal models of polymetastases exist, an oligometastasis(es) model is lacking. Here, we report the first mouse xenograft model of oligometastasis(es) employing the MDA-MB-435 human tumor that maintains a stable oligometastatic phenotype in vivo. We also developed an MDA-MB-435 polymetastatic progression model in which the pattern of dissemination induced poly-foci in the lung, or to multiple anatomic sites including lung, heart, muscle, pleura, bone and peritoneal cavity. We performed microRNA expression profiling from distinct lung metastases of oligometastatic and polymetastatic animals. MicroRNA expression distinguished oligometastatic cell lines from those of polymetastatic, and accurately identified oligometastatic patients from a prior clinical study who failed to develop widespread metastases (p=0.005). These findings form the basis for investigating mechanisms underlying the specific metastatic pattern of individual cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1369. doi:1538-7445.AM2012-1369
Abstract 137: Molecular networks regulated by tumor suppressive microRNA-375 in head and neck squamous cell carcinoma
Authors: Kinoshita Takashi , Hanazawa Toyoyuki , Nohata Nijiro , Kikkawa Naoko , Fuse Miki , Chiyomaru Takeshi , Yoshino Hirofumi , Enokida Hideki , Nakagawa Masayuki , Okamoto Yoshitaka , Seki Naohiko
Published: August 2012

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. In spite of considerable advances in multimodality therapy including surgery, radiotherapy and chemotherapy, the overall five-year survival rate for patients with HNSCC is around 40%. Understanding of the molecular oncogenic pathway underlying HNSCC could significantly improve diagnosis, therapy, and disease prevention. Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma (SCC), maxillary sinus SCC and esophageal SCC revealed that the expression of microRNA-375 (miR-375) was significantly reduced in cancer tissues. In this study, we investigated the functional significance of miR-375 and explored novel molecular networks regulated by miR-375 in HNSCC. METHODS: Cell proliferation assay was performed to investigate the functional significance of miR-375 and its target genes in HNSCC cell lines. Genome-wide gene expression analysis was performed to identify the molecular networks of miR-375 by microarray technique. The expression levels of miR-375 target genes were verified using quantitative real-time RT-PCR in HNSCC clinical specimens. A luciferase reporter assay was used to identify the actual binding site between miR-375 and its candidate target genes. RESULTS: Restoration of miR-375 significantly inhibited cancer cell proliferation in HNSCC cells. Genome-wide molecular targets search and TargetScan database indicated that homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1), lactate dehydrogenase B (LDHB), PC4 and SFRS1 interacting protein 1 (PSIP1), metadherin (MTDH) and solute-carrier family 7, member 11 (SLC7A11) were the candidate genes of miR-375 target. Among them, the expression levels of LDHB and MTDH were significantly up-regulated in clinical HNSCC tumor specimens compared with adjacent normal epithelium. Luciferase reporter assay showed that LDHB and MTDH were directly regulated by miR-375. Silencing of these genes studies demonstrated significant inhibition of cell proliferation in HNSCC cells. CONCLUSIONS: miR-375 functions as a tumor suppressor in HNSCC. LDHB and MTDH are directly regulated by miR-375. These genes may function as oncogenes and contributed to cell proliferation in HNSCC. Tumor suppressive miR-375 and its target oncogenes may provide new insights into the molecular networks of HNSCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 137. doi:1538-7445.AM2012-137
Abstract 1370: Tumor endothelial cells acquire drug resistance by MDR1 upregulation via VEGF signaling in tumor microenvironment
Authors: Akiyama Kosuke , Ohga Noritaka , Hida Yasuhiro , Kawamoto Taisuke , Sadamoto Yoshihiro , Ishikawa Shuhei , Maishi Nako , Akino Tomoshige , Kondoh Miyako , Matsuda Aya , Inoue Nobuo , Shindoh Masanobu , Hida Kyoko
Published: August 2012

Tumor endothelial cells (TECs) are therapeutic targets in antiangiogenic therapy. Contrary to the traditional assumption, it has been reported that TECs can be genetically abnormal and might acquire drug resistance. In this study, mouse TECs and normal ECs (NECs) were isolated to investigate drug resistance of TECs and the mechanism by which it is acquired. TECs were more resistant to paclitaxel with upregulation of multidrug resistance 1 (MDR1) mRNA, which encodes the P-glycoprotein, compared to NECs. Normal human microvascular ECs (HMVECs) were cultured in tumor-conditioned medium (tumor CM) and were found to become more resistant to paclitaxel through MDR1 mRNA upregulation and nuclear translocation of Y-box-binding protein 1 (YB-1), which is an MDR1 transcription factor. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Akt were activated in HMVECs by tumor CM. We observed that tumor CM contained a significantly high level of VEGF. A VEGF receptor kinase inhibitor, Ki8751, and a PI3K/Akt inhibitor, LY294002, blocked tumor CM-induced MDR1 upregulation. MDR1 upregulation via the VEGF/VEGFR pathway in the tumor microenvironment is one of the mechanisms of drug resistance acquired by TECs. We observed that VEGF secreted from tumors upregulated MDR1 through VEGFR2 and Akt activation. This is a novel mechanism of acquisition of drug resistance by TECs in a tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1370. doi:1538-7445.AM2012-1370
Abstract 1373: Preclinical activity of axitinib and its associated change of serum biomarkers in nasopharyngeal carcinoma (NPC)
Authors: Hui Edwin Pun , Lui Vivian WY , Hui Connie WC , Lau Cecilia PY , Loong Herbert HF , Wong Cesar SC , Wong Eric CH , Chan Anthony TC
Published: August 2012

Background: We have previously demonstrated promising activity of sunitinib, a multi-targeted tyrosine kinase inhibitor (TKI) against vascular endothelial growth factor (VEGF) receptor, platelet-derived growth factor receptor (PDGFR), c-kit and RET, in preclinical models of NPC (Invest New Drugs 2011:1123-31). However, the significant host related toxicity encountered in a phase 2 clinical trial of NPC patients has limited its clinical efficacy (Ann Oncol 2011:1280-7). Axitinib is a highly selective TKI of VEGF receptor 1, 2 and 3. Selectively targeting a single growth factor receptor pathway provides the potential to rationally adjust dosages and combine drugs directed at specific parts of the pathway to minimize toxicity and achieve the optimum therapeutic benefit. Methods: In vitro cytotoxicity of axitinib was evaluated by MTT assay in five NPC cell lines (C666-1, CNE-2, HK1-LMP1, HNE-1, HONE-1-EBV). In vivo activity was tested in two representative NPC xenograft models (CNE-2 and HK1-LMP1). We also studied treatment induced changes in tumor histology, microvessel density (MVD) and murine serum biomarkers. Results: All NPC cell lines tested except C666-1 were sensitive to axitinib with IC50 of 0.8-7 µM and maximum growth inhibition of 45-87%. In vivo, axitinib demonstrated significant tumor growth inhibition (fractional tumor volume reduction of 32-63% for axitinib treated mice versus 0-10% for vehicle control), reduced MVD and induced extensive tumor necrosis with no significant host toxicity in mice. There were significant increases in serum murine-derived VEGF and SDF-1α and decrease of sVEGFR-2 in axitinib treated tumor bearing mice as compared to vehicle treated controls. Conclusion: Axitinib demonstrated potent in vitro and in vivo activity in NPC preclinical models. Host derived serum proteins may serve as potential biomarkers of drug activity. A phase 2 clinical trial of axitinib in NPC is on-going (NCT01249547) to validate these findings in patients. Acknowledgement: supported by a direct grant (2041496) from CUHK. Axitinib was provided by Pfizer Inc.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1373. doi:1538-7445.AM2012-1373
Abstract 1374: Cross-resistance and sensitivity of sunitinib resistant tumor cells
Authors: Gotink Kristy J. , Broxterman Henk J. , de Haas Richard R. , Rovithi Maria N. , Pili Roberto , Verheul Henk MW
Published: August 2012

Background: Resistance to sunitinib, a multi-targeted anti-angiogenic tyrosine kinase inhibitor (TKI) approved for clinical use in advanced renal cell cancer, gastrointestinal stromal tumors and pancreatic neuroendocrine tumors, represents a major clinical problem. Previously we have shown that tumor cells acquire resistance in vitro by continuous incubation with sunitinib and found that acidic lysosomal sequestration of sunitinib mediates its resistance. In this study, we investigated whether sunitinib resistant tumor cells are cross-resistant to other clinically relevant inhibitors and whether agents interfering with lysosomal function can modulate resistance. Methods: Continuous exposure to sunitinib for more than 12 months resulted in drug resistance in 786-O (RCC) and HT-29 (colorectal cancer) cell lines. Using these cells, cross-resistance to other clinically available TKIs such as sorafenib, pazopanib and erlotinib, and to the mTOR inhibitor everolimus, was determined in proliferation assays (MTT). In addition, modulation of sunitinib resistance by the lysosomal inhibitors (LIs) chloroquine and bafilomycin A1 was studied using proliferation assays. The effect of LIs on phosphorylation of key signaling proteins was determined by western blotting. Results: Compared to parental cell lines, proliferation assays revealed that sunitinib resistant cell lines were cross-resistant to pazopanib and erlotinib, while no significant decrease in sensitivity to sorafenib or everolimus was observed. Both chloroquine and bafilomycin A1 were able to modulate sunitinib resistance. Western blot analysis revealed that phosphorylation of key signaling proteins including Akt and p44/42-MAPK was inhibited in sunitinib resistant cells upon co-incubation with chloroquine, while sunitinib alone did not inhibit phosphorylation of these kinases. Conclusion: We found that sunitinib resistant tumor cells are cross-resistant to pazopanib and erlotinib, but remain sensitive to sorafenib and everolimus. Furthermore, combination with the lysosomal inhibitor chloroquine resulted in inhibition of key signaling proteins in sunitinib resistant cells. Taken together, we envision that these findings are relevant for clinical treatment strategies in patients with renal cell cancer resistant to sunitinib.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1374. doi:1538-7445.AM2012-1374
Abstract 1375: Contrasting effects of VEGF pathway inhibitors in primary and metastatic disease using perioperative neoadjuvant mouse models
Authors: Ebos John M.L. , Lee Christina R. , Cruz-Munoz William , Jedeszko Christopher , Kerbel Robert S.
Published: August 2012

Recent clinical trial failures with antiangiogenic therapy in patients with early-stage disease have raised the possibility that drug efficacy in preclinical studies, typically involving models of localized primary grown tumors, may not always predict for efficacy in treating metastatic disease, which is the most common cause of mortality in patients. Moreover, depending on disease stage and treatment circumstances, the efficacy of VEGF-pathway inhibitors administered as monotherapies may be offset in certain settings by increased aggressive invasiveness and augmented metastatic potential. Thus, with hundreds of trials underway in neoadjuvant and adjuvant settings to evaluate the efficacy of antiangiogenic therapy in blocking or slowing micrometastatic disease and eventual tumor recurrence, there is an urgent need clarify the utility of such drugs in all stages of tumor progression. Herein we describe a novel preclinical neoadjuvant therapeutic methodology used to compare the effects of short-term VEGF pathway inhibition in highly metastatic human breast, melanoma, and kidney tumor cells grown orthotopically in SCID mice before and after surgical resection. While cytotoxic chemotherapy administered in the maximum tolerated dose could slow tumor growth and lead to a prolongation of survival after resection, similar significant benefits by various VEGF inhibitors in primary tumor inhibition did not consistently translate into significant prevention of recurrent local and distant metastasis after treatment cessation. Furthermore, reductions in primary tumor growth by neutralizing antibodies to VEGF or VEGFR-2 were more predictive of modest survival benefits after tumor resection, compared to the VEGFR tyrosine kinase inhibitor (TKI) sunitinib, which had either no effect or decreased survival compared to control - despite significant reductions in primary tumor growth after neoadjuvant treatment. Potential tumor-independent ‘host’ drug responses could account for this difference as short-term treatment of immunocompromised nu/nu mice prior to intravenous tumor inoculation lead to modest or negligible benefits with VEGF or VEGFR2 antibodies compared to worse outcomes in VEGFR TKI pre-treated animals. Taken together, preclinical neoadjuvant therapy allows for the distinction of ‘anti-primary’ and ‘anti-metastatic’ effects and may serve as a predictive tool to model ongoing clinical trials as well as indicate potential drug combinations which may be used to overcome limitations when such drugs are used as a monotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1375. doi:1538-7445.AM2012-1375
Abstract 1379: The role of PERK and the unfolded protein response in the development of resistance to antiangiogenic agents in renal cell carcinoma
Authors: Zhu Shudong , Panka David J. , Mier James W. , Cho Daniel C.
Published: August 2012

Introduction: Intrinsic and delayed resistance of renal cell carcinoma (RCC) towards agents targeting VEGF-signaling remains a major therapeutic problem. The initial regression of RCC metastases observed in many patients treated with VEGFR2 antagonists is due to the hypoxia and diminished supply of nutrients resulting from the involution of tumor microvasculature. However, hypoxia also elicits a series of adaptive responses in tumor cells that enhance their survival, including activation of the unfolded protein response. To investigate the role of the unfolded protein response in the resistance of RCC to antiangiogenic agents, we assessed the effects of suppressing PERK in RCC cell lines. Experimental Design: A 786-O cell line was generated which was transfected with tetracycline-regulable shRNA targeting PERK. The 786-0 PERK shRNA cell line was exposed to hypoxia (0.5% O2) and glucose deprivation both in the presence and absence of doxycycline. Cells thus treated were lysed after 24 hours for western blot analysis for PERK, LC3, phospho-ACC, BIP, and phopsho-eIF2α. Similarly treated cells were also analyzed after 48 hours for viability by propidium iodide staining. Xenografts were generated from 786-O PERK snRNA cells in nude beige mice and grown to 10mm in size. Mice were then treated with saline, doxycycline (2mg/ml in drinking water), sunitinib (50mg/kg) once daily by gavage, or the combination of both doxycline and sunitinib. Results: Treatment of 786-O PERK shRNA cells with doxycycline suppressed expression level of PERK by approximately 80%. Knockdown of PERK significantly increased cell death resulting from exposure of cell lines to hypoxia and nutrient deprivation in vitro associated with diminished expression of LC3. Treatment of mice bearing 786-0 PERK snRNA xenografts with doxycycline or sunitnib alone had little effect on tumor growth compared with saline treated mice. However, the combination of both sorafenib and doxycline resulted in a sustained tumor growth arrest. Conclusions: These data suggested that PERK and the unfolded protein response have a critical role in the ability of tumor cells to survive the insult of hypoxia and nutrient deprivation caused by the diminished vasculature induced by antiangiogenic therapy. Therapeutic strategies to oppose to unfolded protein response may be promising adjuncts to antiangiogenc agents in RCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1379. doi:1538-7445.AM2012-1379
Abstract 138: The effects of microRNA transfections on global patterns of gene expression are functionally coordinated
Authors: McDonald John F. , Shahab Shubin , Mezencev Roman , Matyunina Lilya , Wang LiJuan
Published: August 2012

A current challenge in the application of miRNAs in cancer therapeutics is the inability to accurately predict the functional consequences of exogenous perturbations in the levels of potentially therapeutic miRNAs. To systematically address this issue, two miRNAs (miR-7 and miR-128) were separately transfected into a well characterized ovarian cancer cell line (HEY) and global changes in levels of gene expression were monitored. While ∼20% of the changes in expression patterns of hundreds to thousands of genes could be attributed to direct miRNA-mRNA interactions, the majority of the changes were indirect, involving the downstream consequences of miRNA-mediated changes in regulatory gene expression. The pathways most significantly affected by miR-7 transfection are involved with cell adhesion and other developmental networks previously associated with epithelial-mesenchymal transitions (EMT) and other processes linked with metastasis. In contrast, the pathways most significantly affected by miR-128 transfection are more focused on cell cycle control and other processes commonly linked with cellular replication. These computational predictions were experimentally verified. Our results indicate that changes in patterns of gene expression induced by exogenous expression of miRNAs are not random but functionally coordinated and that different families of miRNAs may regulate distinct cellular functions related to cancer. Our findings suggest that miRNAs may be of unique therapeutic value by providing clinicians with a strategy to treat cancer from a systems rather than a single gene perspective.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 138. doi:1538-7445.AM2012-138
Abstract 1380: Mechanisms of resistance to antiangiogenic therapy in preclinical models of head and neck squamous cell carcinoma (HNSCC)
Authors: Gyanchandani Rekha , Grandis Jennifer R. , Kim Seungwon
Published: August 2012

Purpose: The Vascular Endothelial Growth Factor (VEGF) is ubiquitously expressed and targeted with FDA-approved agent bevacizumab in many types of cancer including colon, lung and renal carcinoma. In head and neck cancer, bevacizumab is under active clinical investigation. However, its efficacy in most cases is limited, with only modest response rates observed in the clinical trials. These incomplete drug responses suggest the presence of alternative signaling mechanisms that compensate for VEGF blockade. The proinflammatory cytokine, Interleukin 8 (IL-8) is known to promote tumor angiogenesis, and is coexpressed with VEGF in HNSCC. Hence, IL-8 signaling may be a possible mechanism of resistance to anti-VEGF therapy. Experimental Design: In this study, we assessed the efficacy of bevacizumab in preclinical HNSCC models to identify tumors that were sensitive or resistant to the treatment. In order to identify potential mediators of bevacizumab resistance, we evaluated the angiogenic profile of HNSCC cells from the non-isogenic pair of sensitive and resistant cell lines by performing angiogenesis antibody array. We further examined the potential contribution of IL-8 signaling to the in vivo growth of bevacizumab-resistant xenografts, using shRNA-mediated knockdown of the IL-8 gene. Results: HNSCC cell lines showed a range of response to bevacizumab in vivo. Interestingly, the resistant cell lines expressed high levels of proangiogenic factors including IL-8, IL-1a and VEGF compared to the sensitive cell lines. Further, combined inhibition of VEGF and IL-8 in the resistant xenografts resulted in a significant reduction in tumor volume and microvessel density compared to targeting VEGF alone. Conclusions: Our results indicate that HNSCC xenografts display a differential response to anti-VEGF therapy. IL-8 was the most differentially expressed protein in the angiogenic signature of the bevacizumab-resistant and sensitive cells. Finally, abrogation of IL-8 signaling sensitized the resistant xenografts to anti-VEGF therapy. These findings suggest that IL-8 may be associated with anti-VEGF resistance in HNSCC. Hence, co-targeting of VEGF and IL-8 may improve antiangiogenic therapeutic efficacy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1380. doi:1538-7445.AM2012-1380
Abstract 1382: Increased expression of placental growth factor is linked with shortened survival of patients with advanced ovarian cancer
Authors: Block Matthew S. , Maurer Matthew J. , Ramar Priya , Behrens Marshall D. , Erskine Courtney L. , Kalli Kimberly R. , Hartmann Lynn C. , Knutson Keith L.
Published: August 2012

Background: Ovarian cancer (OC) is the leading cause of gynecologic cancer death in the United States. Survival in advanced OC is highly variable; some patients are cured of their disease with a combination of surgery and systemic chemotherapy, while the majority of patients develop recurrence and ultimately die of metastatic OC. While markers of poor prognosis within advanced OC exist, they have not translated to changes in therapy or outcomes. Vascular growth factors are important soluble mediators of tumor growth, vasculogenesis and angiogenesis that may be targeted therapeutically. We therefore explored the association between plasma levels of vascular growth factors and clinical outcomes in advanced OC. Methods: We enrolled 200 patients with OC or benign surgical conditions from the Mayo Ovarian SPORE Biospecimens Core. Presurgical plasma samples from patients with advanced OC (AJCC Stage III and IV, n = 100), early stage OC (Stage I and II, n = 50), and benign surgical conditions (n = 50) were assayed for vascular growth factors via multiplex electrochemiluminescence and by ELISA. Differences in plasma levels between benign, early, and advanced OC patient groups were assessed using plate-adjusted linear models. Associations between plasma levels of vascular growth factors and overall survival in advanced OC patients were assessed by Cox proportional hazards models. Results: Both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) levels were increased in patients with advanced OC (mean ng/mL = 130.8, p = 0.004; mean ng/mL = 20.1, p = 0.025, respectively) compared to patients with early stage (mean ng/mL = 75.1 and 19.0 respectively) and benign conditions (mean ng/mL = 86.8 and 15.0, respectively). An increase in PlGF levels was associated with poor overall survival in patients with advanced OC (HR = 2.52, p = 0.005), while increase in VEGF levels were of borderline significance (HR = 1.21, p = 0.061) Conclusions: Soluble PlGF and VEGF are increased in the setting of advanced OC. Increased plasma PlGF is strongly linked to poor prognosis, while VEGF levels have a borderline association with prognosis. These data provide a rationale for therapeutic targeting of both VEGF and PlGF.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1382. doi:1538-7445.AM2012-1382
Abstract 1383: High levels of CD109+ circulating endothelial cells and progenitors as marker of response to bevacizumab and irinotecan in high grade gliomas
Authors: Cuppini Lucia , Pellegatta Serena , Calleri Angelica , Bruzzone Maria Grazia , Prodi Elena , Anghileri Elena , Mancuso Patrizia , Bertolini Francesco , Finocchiaro Gaetano , Eoli Marica
Published: August 2012

Purpose: Bevacizumab, an anti-VEGF antibody, has shown significant activity in high grade gliomas (HGG). Previous studies emphasized the need for predictive markers of response. Experimental Design: We treated 63 recurrent HGG patients with poor prognostic factors with bevacizumab (10 mg/kg) and irinotecan (125 or 340 mg/m2) every 2 weeks, and investigated the predictive potential of circulating endothelial cells (CECs) and their progenitors (CEPs). Results: After a median follow-up of 27 weeks, median OS and PFS were 33 and 18 weeks, respectively. PFS at 6 and 12 months were 32% and 12%. OS at 6 months was 60%. No complete response but fourteen partial responses according to RANO criteria were observed. Toxicity and side effects were mild. Patients with distant intracerebral disease or leptomeningeal dissemination at baseline MRI had shorter PFS (p=0.002; p=0.01) and OS (p=0.005; p=0.03). Baseline CEP over 32.8 cells/ml (1st quartile) or CD45dimCD34+CD133+ hematopoietic committed progenitors over 27 cells/ml (1st quartile) were associated with an increased PFS (p=0.01; p=0.001, respectively). Baseline CD109+ CECs over 47.5 cells/ml (2nd quartile) were associated with longer PFS and OS (p= 0.001; p=0.02). Patients who progressed after 18 weeks of therapy or more (n=22) had baseline levels of CD109+ CECs and CD45dimCD34+VEGFR2+ cells significantly higher than others (p=0.008; p=0.04, respectively). At progression no significant change in CEC and CEP was detected. Conclusions: The data point to baseline CD109+ CECs, CEP and CD45dimCD34+CD133+ hematopoietic committed progenitors as promising markers for the selection of patients who could benefit from bevacizumab in the treatment of HGG.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1383. doi:1538-7445.AM2012-1383
Abstract 1384: Axl and VE-cadherin expression as markers of angiogenesis in human breast cancers
Authors: Veien Kristi Anne , Krüger Kristi , Stefansson Ingunn , Arnes Jarle B. , Collett Karin , Akslen Lars A.
Published: August 2012

Background: The aim of our study was to investigate vascular expression of Axl and VE-cadherin as potential markers of angiogenesis in human breast carcinoma. Materials and methods: Immunohistochemical staining was performed using antibodies against Axl and VE-cadherin. Expression was examined in a population-based series of breast cancers collected in Hordaland County (Norway) during 1996-2001. The series is a nested case-control study as part of the Norwegian Breast Cancer Screening Program, and consists of 95 invasive interval cancers and 95 invasive screen detected cancers matched by tumor diameter. Positive microvessels were counted in 10 consecutive high power fields within hot-spot areas according to previously established criteria and expressed as microvessel density (MVD) by mean number of microvessels per mm2. Results: The MVD for Axl was associated with progesterone receptor status (p=0.026), HER-2 status (p=0.030) and basal-like phenotypes: CK5/6+ P-cadherin+ (p=0.009), ER- HER2-CK5/6+ (p=0.014) and ER- HER2- CK5/6+ and/or EGFR+ (p=0.027). In contrast, MVD by VE-cadherin expression (n=127) indicated negative associations with basal-like phenotypes: CK5/6+ or P-cadherin+ or EGFR+ (p=0.034), and ER- HER2- P-cadherin+ (p=0.003). VE-cadherin expression also tended to be associated with better patient survival (p=0.067). Conclusion: High MVD by vascular expression of Axl was associated with aggressive features in our breast cancer series, especially the basal-like phenotype, whereas MVD by VE-cadherin expression appeared to be related to less aggressive tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1384. doi:1538-7445.AM2012-1384
Abstract 1390: In vivo tracking of cancer-cell-derived exosomes
Authors: Homma Kimi , Suetsugu Atsushi , Ochiya Takahiro , Hoffman Robert M.
Published: August 2012

The goal of the present study was to determine if exosomes play a role in cell-to-cell communication to promote tumor metastasis. Green fluorescence protein (GFP) was linked to CD63, which is a general marker of exosomes, in order to track breast-cancer-cell-derived exosomes cancer in nude mice. Breast cancer cells transferred their own exosomes to other cancer cells and normal lung tissue cells in culture. In the nude mouse, breast cancer cells secreted GFP exosomes into the tumor micro environments as well as the circulation. Numerous GFP in exosomes were visualized in breast cancer metastases in nude mice. The results of this study suggest that tumor-derived exosomes may contribute to formation of a niche to promote metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1390. doi:1538-7445.AM2012-1390
Abstract 1391: Breast cancer cells and the bone: A tale of treachery
Authors: Das Shamik , Tucker J. Allan , Khullar Shikha , Samant Rajeev S. , Shevde Lalita A.
Published: August 2012

Under normal physiological conditions there is a dynamic equilibrium in the bone maintained by the bone forming osteoblasts and bone resorbing osteoclasts. The bone stroma provides hospitable sites for disseminating, colonization-competent breast cancer cells. The Hedgehog (Hh) pathway plays an important role in skeletogenesis. Our studies have determined that breast tumor cells have an activated Hh pathway characterized by upregulation of Hh ligands and the transcription factor GLI1. Our findings show that breast cancer cells activate Hh signaling in pre-osteoclasts and thereby upregulate osteoclastogenesis. Paracrine Hh signaling induced in osteoclasts by breast cancer cells mediates a series of events by the up-regulation of osteopontin (OPN), which in turn enhances osteoclastic activity by up-regulating the essential proteases cathepsin K and MMP9. Surprisingly, we found that breast cancer cells also activate osteoblast differentiation as an early event, by activating Hh signaling in pre-osteoblasts. This leads to the expression of RANKL and PTHrP by the osteoblasts. RANKL and PTHrP promote osteoclastogenesis while the osteoblasts are eliminated by apoptosis. Pharmacological inhibition of Hh signaling results in an overall decrease in osteoblast differentiation and osteoclastogenesis/resorptive activity. Thus our studies suggest that the breast cancer cells orchestrate a chain of events in which they directly foster osteoclastogenesis and deceptively induce osteoblasts to differentiate which in turn produce factors that promote osteoclast differentiation. A vicious cycle ensues and in the presence of increased number of metastasing breast cancer cells the balance now shifts in favor of the osteoclasts resulting in osteolysis, the final painful outcome of a majority of breast cancer patients. Thus in the light of our findings, any therapeutic approach utilizing inhibitors for the Hh pathway need to take into consideration the dual role of the pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1391. doi:1538-7445.AM2012-1391
Abstract 1392: Differential effects of Cancer Stem Cells (CSCs) and non-CSCs-targeted therapies on lung primary tumors and metastasis
Authors: Larzabal Leyre
Published: August 2012

Cancer stem cells (CSCs) are thought to be responsible for tumor initiation and recurrence after chemotherapy. Targeting CSCs and non-CSCs with specific compounds may be an effective approach to reduce lung cancer growth and metastasis. Salinomycin was recently identified as a selective inhibitor of CSCs. The aim of this study was to investigate the effect of salinomycin with or without combination with paclitaxel in the Lewis lung carcinoma (LLC) metastatic model. Salinomycin reduced the proportion of ALDH+ CSCs in LLC cells, whereas paclitaxel increased such population. The same effect was observed for the human H460, H1299 and H272 lung cancer cell lines. Salinomycin reduced the tumorsphere formation capacity of LLC by more than 7-fold, but paclitaxel showed no effect. For in vivo experiments, LLC cells were injected subcutaneously and, after surgical removal of the primary tumors, animals were checked for lung metastasis. Paclitaxel reduced primary tumor volume but increased the number of metastatic nodules (p<0.05), whereas salinomycin had no effect on primary tumors but reduced metastasis (p<0.05). Combination of both drugs did not improve the effect of single therapies. mRNA levels of ALDH, CXCR4 and SDF-1 were higher in metastatic lesions than in primary tumors, and were significantly elevated in both locations by paclitaxel treatment. On the contrary, such levels were reduced (or in some cases did not change) when mice were administered with salinomycin. The number of F4/80+ (macrophages) was also decreased upon administration of paclitaxel or salinomycin in both primary tumors and metastasis. The number of mast cells was only reduced in primary and metastatic lesions of paclitaxel-treated mice. These results show that salinomycin targets ALDH+ lung CSCs, which has important therapeutic effects in vivo by reducing metastatic lesions. On the contrary, paclitaxel (although reducing primary tumor growth) promotes the selection of ALDH+ cells that likely modify the lung microenvironment to foster metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1392. doi:1538-7445.AM2012-1392