bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4238: Azadirachta indica leaf extract inhibits mammary carcinogenesis by altering key signaling pathways
Authors: Agullo Pamela A. , Arumugam Arunkumar , Parada Jacqueline , Gamez Monica , Boopalan Thiyagarajan , Delgado Alejandro , Lakshmanaswamy Rajkumar
Published: June 2012

It is estimated that 20-60% of cancer patients utilize some form of alternative medicine. Plant products play a major role in complimentary medicine. Azadirachta indica tree commonly known as neem has been known for centuries to possess medicinal properties like immunomodulatory, anti-inflammatory, antimutagenic, and anticarcinogenic. We have previously shown that A. indica leaf extract has potent anticarcinogenic capabilities in N-methyl-N-nitrosurea (MNU)-induced mammary cancer model. The aim of our study is to explore the molecular pathways through which A. indica induces mammary tumor regression. Seven week old female Lewis rats were administered MNU. On the appearance of the first palpable tumor the rats were divided into groups receiving different doses of ethanolic A. indica leaf extract (ALE): group 1 control, no treatment; group 2 0.125mg/day; group 3: 0.250mg/day; group 4: 0.500mg/day; group 5: 1mg/day. Treatment was daily for 4 weeks intraperitoneally. The animals were euthanized 12 weeks after the termination of treatment and mammary tumor tissue samples were snap frozen and also fixed in formalin. To analyze key gene expression changes we performed pathway focused PCR microarrays for apoptosis, PI3-Kinase, and angiogenesis signaling pathways. Our results show that ALE induces tumor regression, decreases tumor volume, and inhibits mammary tumor growth by altering specific molecular pathways, linked to the stimulation (Bax, caspases) and inhibition (Bcl-2, CDC42)of apoptosis, up-regulation of tumor suppressor genes (PTEN), down-regulation of proto-oncogene Jun, and inhibition of cell survival (Foxg1, BTK, Akt). ALE treatment down-regulated genes fundamental to migration, adhesion and proliferation of tumor cells (EFNA1, Hif1a). Timp1 (TIMP family matrix metalloproteinase inhibitor) and cyclin D1 expression exhibited a clear dose response. Our data indicates that ALE treatment inhibits mammary tumor development by altering apoptosis, PI3-Kinase and angiogenesis pathways. In conclusion, ALE could be used as a novel complimentary agent that offers anticarcinogenic effects against breast cancer and could also be used along with the current standard treatment options.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4238. doi:10.1158/1538-7445.AM2011-4238
Abstract 4244: TTAC-0001, fully human anti-VEGFR2/KDR neutralizing antibody blocks tumor angiogenesis and tumor growth
Authors: Lee Weon Sup , Pyun Bo-Jeong , Kim Sung-Woo , Shim Sang Ryeol , Yoo Hyeon-Mi , Oh Keunhee , Lee Dong-Sup , Yoo Ji-Young , Yun Chae-Ok , Kwon Young-Guen , Lee Sang Hoon , Yoo Jin-San
Published: June 2012

Angiogenesis, the recruitment of new blood vessels is a crucial mechanism required for both tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptors, particularly VEGF receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in tumor-associated angiogenesis. Monoclonal antibodies (mAb), due to their high specificity towards a given target, represent a unique class of novel therapeutics as angiogenesis inhibitors. We developed TTAC-0001, anti-KDR antibody from a fully human naïve single chain variable fragment (ScFv) phage library. Interestingly, TTAC-0001 displayed cross reactivity against murine homologue, Flk-1, which allows us to evaluate the anti-tumor efficacy in various in vivo models. TTAC-0001 inhibits VEGF-mediated proliferation and migration. In HUVEC, TTAC-0001 inhibited the phosphorylation of VEGFR2/KDR and ERK mediated by VEGF. TTAC-0001 inhibited VEGF-mediated sprouting endothelial cells from rat aortic rings and showed a potent anti-angiogenic activity in VEGF-mediated matrigel model in nude mice. Further in vivo anti-tumor efficacy of TTAC-0001 was seen in the xenografts of A549 (lung) and HCT116 (colorectal) human cancer. These changes were accompanied by the inhibition of microvessel density and induction of apoptosis in the tumors. In summary, our data indicates that TTAC-0001 blocks the binding of VEGF to VEGFR2/KDR and inhibits VEGFR-induced angiogenesis. Therefore, this data strongly supports for the further development of TTAC-0001 as an anti-cancer agent (This study was supported by grants of the Korea Healthcare technology R&D Project, Ministry for Health & Welfare Affairs (A092255 and A040016) and Advanced Medi-cluster R&D Project, Daejeon TechnoPark, Republic of Korea).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2011-4244
Abstract 4327: Fasting enhances the response of Gl26-glioma to radiation and multiple chemotherapies
Authors: Safdie Fernando , Brandhorst Sebastian , Wei Min , Wang Weijun , Lee Changhan , Hwang Saewon , Chen Thomas , Longo Valter D.
Published: June 2012

Malignant gliomas like anaplastic astrocytoma and glioblastoma mutliforme (GBM) are among the most commonly diagnosed malignant adult brain tumors. GBM is highly invasive and angiogenic and causes mortality rates higher than any other brain tumor, resulting in a median survival for GBM patients that is ∼12 to 15 months. Treatment of GBM is multimodal, usually initiated by surgical resection, followed by the adjuvant therapy of radiation and chemotherapy. Temozolomide is the current standard of care for patients with newly diagnosed gliomas and is well tolerated. Temozolomide has been shown to delay tumor-progression and prolong patient survival. As the treatment of GBM remains elusive, the improvement of existing therapies becomes important.

The beneficial effects of dietary restriction (DR) on reducing age-dependent diseases, such as cancer, are well established. However, DR is expected to require weeks or even months for efficacy in human patients, which eventually would prove ineffective for cancer patients with fast progressing disease status.

We have previously reported that fasting, an intense but brief form of DR differentially protects normal, as opposed to various cancer cells, against the toxicity of high-dose chemotherapy in in-vitro and in-vivo in a neuroblastoma-bearing mice model by causing major changes including the reduction of glucose, and IGF-I. We termed this phenomenon Differential Stress Resistance (DSR).

We show that mimicking plasma-fasting conditions in-vitro sensitizes Gl26-glioma cells to chemotherapy treatment by Temozolomide and causes enhanced cell death of the starved glioma cells. We than applied this model of sensitization of cancer cells under fasting conditions to an intracranial and a subcutaneous glioma-tumor model in-vivo. We demonstrate that short term fasting enhances chemo- and radiotherapy in the in-vivo model and prolongs survival of tumor bearing mice even when compared to Temozolomide treated animals.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2011-4327
Abstract 433: Altered expression of lumican in prostate cancer is identified by microarray analysis
Authors: Wang Yi , De Velasco Marco A. , Okazaki Erina , Izumi Ayaka , Tanka Motoyoshi , Yamamoto Yutaka , Hatanaka Yuji , Yoshimura Kazuhiro , Nozawa Masahiro , Rosser Charles , Goodison Steve , Yoshikawa Kazuhiro , Nishio Kazuto , Uemura Hirotugu
Published: June 2012

Prostate cancer is one of the principal medical problems facing men worldwide; fortunately mortality rates from prostate cancer have been decreasing in many developed countries, as a result of improved treatments and early detection. Developing biomarkers that correlate to prostate cancer will facilitate the delivery and development of treatment therapies for patients. To accomplish this we performed a comprehensive genome-wide microarray analysis of genes differentially up- or down-regulated in PSACre;PtenloxP/loxP mutant mice and examined novel differentially expressed genes in to identify novel genes that could potentially serve as biomarkers for disease tumor characterization. Total RNAs were collected form the prostate glands of tumor bearing PSACre;PtenloxP/loxP mutant and wild type mice. cDNAs were generated and were then hybridized onto Affymetrix microarray chips and analyzed with Agilent's Gene Spring software. Microarray analysis revealed that lumican was significantly upregulated in Pten mutant mice compared to wildtype controls. qRT-PCR validation was performed and correlated to the microarray data. Immunohistochemical staining was performed on mouse prostates at various stages of the disease process. Two staining patterns were observed and tabulated, 1) prostate epithelium and 2) stroma. Mouse epithelial cell tended to stain negative in normal glands. Expression increased in moderate and poorly differentiated tumor. The highest expression patterns were observed in the prostates from castrate resistant tumors. More notable was the increased an intense staining seen in the stromal component, especially in the thickened basement membranes cancerous glands. We also examined the expression patterns of lumican in 10 cases of human prostate cancer and benign prostatic hyperplasia (BPH). Epithelial expression in human was moderate to high in benign prostatic hyperplasia, Gleason score 6, 9 and metastatic tumors and moderate in Gleason score 7 tumors. Stromal staining was low in BPH and gradually increased to moderate as the Gleason score increased and high in metastatic lesions. The staining pattern of lumican was very similar to that of vimentin in both human and mouse tissues. Our data has shown that lumican may be differentially expressed in various stages of prostate cancer, however, larger number of clinical need to be evaluated to establish a clear correlation to clinicopathological features.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2011-433
Abstract 4334: Targeting p53 null neuroblastomas through RLIP76
Authors: Vatsyayan Rit , Singhal Jyotsana , Yadav Sushma , Nagaprashantha Lokesh Dalasanur , Singhal Sharad S. , Awasthi Sanjay
Published: June 2012

The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53 mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53 mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of pro-apoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione-conjugate transporter, RLIP76. The RLIP76-p53 complex was demonstrated both by using immuno-precipitation analyses of purified proteins as well as by immuno-fluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα stimulated transport of glutathione-conjugates of 4HNE (GS-HNE) and cisplatin. Drug resistance was significantly greater for p53 mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53 mutant neuroblastoma, based on a model of regulation of p53 induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in over-expression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53 mutant neuroblastoma. (Supported in part by NIH grant CA 77495 (SA), CA 155350, Cancer Research Foundation of North Texas, and Institute for Cancer Research & the Joe & Jessie Crump Fund for Medical Education (SSS))

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2011-4334
Abstract 4336: Oncolytic reovirus as a novel therapy for neuroblastoma
Authors: Kellar Amelia , Redding Nicole , Blote Karen , Shi Qiao , Spurrell Jason , Beaudry Paul , Morris Don
Published: June 2012

Background: Neuroblastoma (NB), a tumour of neurocrest progenitor cells of the sympathetic nervous system, is the most common extracranial pediatric tumour and accounts for approximately 15 percent of childhood cancer mortality. Even with surgery, radiation therapy, aggressive cytotoxic chemotherapy including autologous stem cell transplantation and more recently immunotherapy, high risk neuroblastoma has only a 30 percent predicted survival rate. Reovirus, a double-stranded RNA virus, has been shown to be effective against a myriad of cancers through its ability to preferentially lyse cancer cells with aberrant Ras pathway signaling. In this study, we investigate the potential of reovirus (serotype 3, strain Dearing) as a novel treatment for neuroblastoma and neuroblastoma tumour initiating cells (nbTIC).

Experimental Design/Results: Reovirus induces dramatic cytotoxic effects at a multiplicity of infection (MOI) of 40 within 48h as assessed by WST-1 viability assays for the IMR-32, IMR-5, SK-N-AS, SK-N-SH, LAN-1, LAN-5, and SHEP human neuroblastoma cell lines in vitro. Interestingly the human neuroblastoma tumor-initiating cell lines, NB-12, NB88 and NB122 (kind gift from Dr David Kaplan) were also found to be very sensitive to reovirus-induced cytotoxicity, suggesting a role for reovirus treatment of refractory neuroblastoma. In order to further study reovirus, immunotherapy and neuroblastoma, a syngeneic, immunocompetent murine model (Neuro2a, A/J neuroblastoma model) was utilized to test reovirus-induced cytotoxicity and RV-directed immunotherapy of Neuro2A in vitro and in vivo. Preliminary data suggest that the Neuro2a cells are exquisitely sensitive to reovirus in vitro. Updated results will be presented.

Conclusion: These preclinical results suggest that reovirus holds promise as a novel therapeutic for neuroblastoma and that it warrants further investigation in early phase clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4336. doi:10.1158/1538-7445.AM2011-4336
Abstract 4338: The effect of opposing roles of NF-kB on TRAIL-R2 expression in TRAIL cytotoxicity in neuroblastoma cells
Authors: Jung Hye Lim , Lee Myoung Woo , Kim Hye Ryung , Kim Dae Sung , Kim Hye Jin , Lee Soo Hyun , Chueh Hee Won , Yoo Keon Hee , Sung Ki Woong , Koo Hong Hoe
Published: June 2012

TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-kappa B (NF-kB) has been implicated, but its exact role is controversial. In this study, we have investigated the role of NF-kB on enhancement of TRAIL cytotoxicity in neuroblastoma cells. IkB kinase (IKK) inhibitor decreased basal NF-kB activity and marginally induced cell death in neuroblastoma cell line SK-NMC. Pretreatment with IKK inhibitor synergistically increased TRAIL-mediated apoptosis via up-regulation of TRAIL- receptor2 (TRAIL-R2) expression in response to the decrease in basal NF-kB activity, indicating that basal NF-kB activity blocks TRAIL-R2 expression in SK-NMC cells. In addition, pretreatment with Cisplatin, a platinum analog and an alkylating agent widely used in neuroblstomas, showed increase in NF-kB activity and induction of apoptotic cell death in SK-NMC cells. Pretreatment with Cisplatin significantly increased TRAIL-mediated apoptosis via up-regulation of TRAIL-R2 expression through induction of NF-kB, suggesting that induced NF-kB is involved in TRAIL-R2 expression up-regulated by Cisplatin. IKK inhibitor or Cisplatin followed by TRAIL treatment increased caspase-8, -9, and -3 activation. Mcl-1 cleavage and Bid truncation. Our results implicate that basal NF-kB activity is involved in TRAIL resistance but induced NF-kB plays a role in TRAIL sensitivity, and suggest that the enhanced sensitivity of SK-NMC cells to TRAIL is associated with an increase in TRAIL-R2 expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4338. doi:10.1158/1538-7445.AM2011-4338
Abstract 4339: Evaluation of polyamine inhibitors in N/S/I-type neuroblastoma and hypoxia
Authors: Bachmann Andre S. , Borsics Tamas
Published: June 2012

Background: Neuroblastoma (NB) is the most common extracranial childhood tumor and is derived from neural crest cells of the sympathetic nervous system. There is currently no curative treatment for children with relapsed/refractory NB, and for these children the 5-year survival rate is <10%. As such, new therapeutic approaches with alternative modes of action are needed for treatment. Since MYCN controls a number of genes including ornithine decarboxylase (ODC), we proposed that ODC should be considered as a novel target for MYC-driven tumors such as NB (Oncogene, 2005, 24:5606-18; Int J Cancer, 2010, 126:2012-24). Our previous research identified two polyamine inhibitors, DFMO and SAM486A, as effective agents for the treatment of NB (Cancer Res, 2008, 68:9825-31; Mol Cancer Ther, 2009, 8:2067-75). Methods: We utilized in vitro MTS cell viability testing to assess the differential effects of DFMO and SAM486A in three specific NB cell sub-populations (neuroblastic (N), Schwann cell-like/adherent (S), and an intermediate (I) morphology types) in the presence or absence of functional tumor suppressor p53. A hypoxia chamber, Western blot analysis, and microscopy were applied to analyze the effect of polyamine inhibitors in a hypoxic environment. Results: While weak apoptosis was induced by DFMO in all three cell types with non-functional p53, SAM486A was most potent in N-type NB cells with functional p53. Interestingly, the potency of SAM486A in p53 wt cells under hypoxic conditions was significantly reduced. Conclusion: Our data provide evidence that the p53 status, NB cell sub-type and the presence/absence of oxygen in the tumor cell environment are crucial factors and determine the overall sensitivity of individual polyamine inhibitors. These new insights are important as polyamine metabolism-targeted NB therapy with DFMO has now entered the clinical stage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4339. doi:10.1158/1538-7445.AM2011-4339
Abstract 4341: Bortezomib primes neuroblastoma cells for TRAIL- triggered apoptosis by linking the death receptor to the mitochondrial pathway
Authors: Naumann Ivonne , Kappler Roland , von Schweinitz Dietrich , Debatin Klaus-Michael , Fulda Simone
Published: June 2012

In contrast to remarkable improvements in the survival and cure rates that have been encountered for many childhood malignancies, the prognosis of children with advanced stage neuroblastoma remains poor.

Searching for novel strategies to modulate apoptosis in neuroblastoma we investigated the potential of the proteasome inhibitor Bortezomib to modulate TRAIL-induced apoptosis in neuroblastoma cell lines, primary neuroblastoma cultures and in an in vivo model of neuroblastoma.

Here, we provide first evidence that Bortezomib at subtoxic concentrations synergistically cooperates with TRAIL to induce apoptosis (combination index 0.5). In addition, Bortezomib acts in concert with TRAIL to reduce colony formation of neuroblastoma cells, demonstrating an effect also on longterm survival. Mechanistic studies reveal that Bortezomib profoundly enhances TRAIL-induced cleavage of Bid into tBid, accumulation of tBid in the cytosol and its insertion into mitochondrial membranes, pointing to a concerted effect on Bid cleavage (TRAIL) and stabilization of tBid (Bortezomib), which links the death receptor to the mitochondrial pathway. Additionally, Bortezomib increases expression of p53 and Noxa. All these changes lead to increased activation of Bax and Bak, loss of the mitochondrial membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis upon treatment with Bortezomib and TRAIL. Knockdown of either Bid, Noxa or p53 significantly delays the kinetic of Bortezomib- and TRAIL-induced apoptosis, whereas it does not confer longterm protection. By comparison, overexpression of Bcl-2, which simultaneously antagonizes tBid, Noxa and p53, significantly inhibits Bortezomib- and TRAIL-induced apoptosis and even rescues clonogenic survival. Of note, Bortezomib and TRAIL act in concert to trigger apoptosis in several patients’ derived primary neuroblastoma cultures, underscoring the clinical relevance. Importantly, Bortezomib cooperates with TRAIL to suppress tumor growth in an in vivo model of neuroblastoma.

In conclusion, these findings demonstrate for the first time that Bortezomib at subtoxic concentrations represents a promising new approach to prime neuroblastoma cells towards TRAIL-induced apoptosis by linking the death receptor to the mitochondrial pathway. Thus, this study provides the rational for future (pre)clinical evaluation of the combination strategy with Bortezomib and TRAIL in neuroblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4341. doi:10.1158/1538-7445.AM2011-4341
Abstract 4342: The allosteric Akt inhibitor MK-2206 inhibits neuroblastoma tumor cell growth in vitro and in vivo
Authors: Li Zhijie , Ramalingam Sridevi , Yan Shuang , Thiele Carol J.
Published: June 2012

Background: Activated Akt in tumor tissue of NB patients is a marker of decreased event-free as well as overall survival. Our studies indicate activated Akt induces chemoresistance in NB cells and chemoresistance is a major problem in the treatment of Neuroblastoma (NB). The allosteric Akt inhibitor, MK-2206 inhibits the activation of the 3 Akt isoforms with IC50-Akt1 = 5.3nM, -Akt2 = 12nM and -Akt3 = 65nM. In this study we tested the effects of MK-2206 in vitro and in vivo pre-clinical models of NB.

Methods: NB cell lines (NGP, SY5Y, KCNR, BE2, SKNFI, and AS) were treated with increasing concentrations of MK-2206 for 48 hours. MK-2206-induced cell death was evaluated by MTS assay. Phosphorylated Akt and its downstream target S6 were determined by Western Blot analysis. The effect of MK-2206 (100 or 200mg/kg/3 days/wk) on NB tumor growth was evaluated in a xenograft nude mouse model.

Results: NB cell lines express all 3 Akt isoforms to varying levels. In in vitro studies, MK-2206 inhibited cell growth in all NB cell lines although with different sensitivities. The growth of NGP, SY5Y, KCNR and BE2 cells were more sensitive to MK-2206, with IC50<4μM; while the growth of AS and SKNFI were less sensitive, with IC50>11μM. MK-2206 treatment decreased phosphorylated-Akt and its target phosphorylated-S6 in NB cells to a comparable level in all tested cell lines regardless of biologic sensitivity to MK-2206. In in vivo studies, mice bearing the sensitive BE2 NB tumors were treated with MK-2206 (100 or 200mg/kg) while mice bearing the less sensitive AS tumors received MK-2206 (200mg/kg). There was an approx. 30% inhibition of tumor growth in each tumor model only in mice treated with 200mg/kg MK-2206 (P<0.01). MK-2206 (200mg/kg) treatment led to a comparable increase in survival in both models (P<0.01).

Conclusion: This study indicates that despite differences in in vitro growth sensitivities, the Akt inhibitor, MK-2206 has a comparable anti-tumor cell growth inhibiting activity in vivo. This study supports the future evaluation of the combination of MK-2206 with chemotherapeutic drugs to improve the treatment efficacy in NB.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4342. doi:10.1158/1538-7445.AM2011-4342
Abstract 4347: Anaplastic lymphoma kinase (ALK) expression is an independent prognostic factor in neuroblastoma patients and correlates well with ALK inhibitor response in vitro
Authors: Duijkers Floor A.M. , Gaal Jose , Meijerink Jules P.P. , Admiraal Pieter , Pieters Rob , de Krijger Ronald R. , van Noesel Max M.
Published: June 2012

Introduction: ALK mutations are one of the most common genetic aberrations occurring in 3-11% of the neuroblastoma (NBL) patients and are considered oncogenic. We determined the role of ALK protein levels on survival of NBL patients. Secondly, we tested in vitro responsiveness to ALK inhibitors in NBL cell lines with and without ALK mutation.

Results: Formalin-Fixed, Paraffin-Embedded material of 71 NBL, 12 ganglioneuroblastoma (GNBL) and 20 ganglioneuroma (GN) patients was stained for total ALK protein by immunohistochemistry (IHC). The percentage of ALK positive cells in IHC was stratified in 4 Ccategories; Category 1 (n=40): < 20% ALK-positive cells, category 2 (n=25): 20-50% ALK positive cells, category 3 (n=25): 50-75% ALK positive cells, and category 4 (n=13): 75-100% ALK positive cells. These categories correlate significantly with overall survival in the spectrum of NBL, GNBL and GN (p=0.002, Logrank test). ALK mutations were identified in 2/63 NBL (3.2%) and 2/11 GNBL (18.2%) patients by sequencing. Importantly, in NBL and GNBL patients, ALK protein expression remains a significant factor in a multivariate model (Cox Proportional Hazard model) including the COG risk stratification (p<0.000), which encompasses stage and NMYC status (see table).

Further, we studied the role of ALK levels in the response to ALK inhibitors in vitro in NBL cell lines. ALK inhibitor response (TAE684) correlates well with ALK protein levels (r=−0.813, p=0.001). ALK point mutated cell lines show higher ALK levels and a better ALK inhibitor response (LC50 values 14.9 fold lower, p=0.004) than WT and amplified cell lines.

In conclusion: ALK protein expression is an independent predictor of overall survival in NBL patients and ALK inhibitor response in vitro. ICH staining with ALK antibody is reliable and correlates well with prognosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4347. doi:10.1158/1538-7445.AM2011-4347
Abstract 4349: The tumor suppressor WTX is inactivated in a subset of pediatric tumors
Authors: Akhavanfard Sara , Rivera Miguel N. , Dias-Santagata Dora C. , Vargas Sara O. , Pomeroy Scott , Haber Daniel A. , Iafrate A. John
Published: June 2012

WTX is a recently identified tumor suppressor gene that is expressed during embryonic kidney development and that is inactivated by deletions and truncations in approximately 30% of Wilms tumors, the most common pediatric kidney cancer. Initial functional studies point to a role for this gene in Wnt signaling and WT1 mediated transcription. Given that WTX is also present in a variety of other tissues during development, we tested for potential involvement of WTX in other pediatric tumors. 25 hepatoblastomas, 87 rhabdomyosarcomas, 11 adrenocortical carcinomas, 15 pleuropulmonary blastomas, 30 neuroblastomas and 30 medulloblastomas were tested by 3 color- fluorescence in situ hybridization (FISH) with probes corresponding to WTX (Xq11.1) and two flanking control sites. Discrete WTX deletions were identified in two cases, one hepatoblastoma and one rhabdomyosarcoma, and not in matched control tissues. Direct sequencing of the full WTX open reading frame in 24 hepatoblastomas, 27 rhabdomyosarcomas and 11 adrenocortical carcinomas did not reveal point mutations in these tumor types. CTNNB1 exon 3 mutations, which are present in Wilms tumor but generally do not overlap with WTX inactivation, were detected in 8 of 25 hepatoblastomas but not in the tumor with a WTX deletion. Our results indicate that WTX inactivation is a genetic event that occurs in other pediatric malignancies, albeit at a lower frequency than in Wilms tumors. Notably, tumors with evidence of WTX inactivation, Wilms tumor, hepatoblastoma and rhabdomyosarcoma, constitute a subset of pediatric tumors that are linked to abnormalities of the IGF2 locus on 11p15 in the Beckwith-Wiedemann overgrowth syndrome. Finally, the high frequency of WTX inactivation in Wilms tumors compared to other pediatric malignancies, points to the specificity of the connection between WTX and the kidney developmental pathways that are dysregulated in this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4349. doi:10.1158/1538-7445.AM2011-4349
Abstract 4353: Primary sarcoma of the liver and biliary tract in children: Results for the International Society of Paediatric Oncology (SIOP) studies MMT 84, MMT89 and MMT95
Authors: van Noesel Max M. , Mulder Willemijn , Rey Annie , Kelsey Anna , Stevens Michael C.G. , Martelli Hélène , Oberlin Odile
Published: June 2012

Purpose: Sarcomas of the liver (SL) are mostly Undifferentiated Embryonal Sarcoma of the Liver (UESL), or RMS. Sarcoma of the biliary tract (SBT) are usually botryoid or embryonal RMS. The prognosis is not well known. The value of common prognostic factors, (histology, tumor size, age, IRS grouping, chemotherapy) have only been described in case reports or very limited series.

Patients and Methods: Here, we analyzed outcome and modality of relapse for patients under 18 years of age with nonmetastatic SL and SBT of the prospective SIOP Malignant Mesenchymal Tumors studies (MMT 84, 89 and 95). MMT studies are based on IVA (Ifosfamide, vincristine, actinomycin-D) and local surgery and/or radiotherapy.

Results: The MMT studies (1984-2003) included 1554 sarcoma patients, of which 39 SL (11 RMS and 28 UESL) and 8 SBT, total 47 (3%). The median age was 6.3 years, M:F=39:10. The 5 years OS and EFS were 80%, not significantly different in RMS vs. UESL, or liver vs. biliary tract. Pre-surgical T1 vs. T2 tumors were significantly different in prognosis (5-yrs EFS 85 % vs. 62 %, resp.; p=0.03), but this was non-significantly different in the post surgical IRS staging (group I, II vs. III; p=0.08). Age at diagnosis, tumor size or histology, and treatment protocol were none significantly related to outcome. Complete remission (CR) was achieved in 45/47of the patients (96%), 7/47 (15%) after primary surgery, 3/47 after chemotherapy, 31/45 after surgery and chemo, 4/47 after chemo, radiotherapy and surgery, In 22 second, delayed surgery was performed, 21/22 achieved CR. Relapsed patients (11/47; 23%) did not survive. The effect of chemotherapy was no different between studies (MMT 84, 89, 95).

Conclusion: In the SIOP MMT studies, an IVA based chemotherapy combined with local treatment resulted in a favorable outcome for sarcomas of the liver and bile duct.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4353. doi:10.1158/1538-7445.AM2011-4353
Abstract 4354: Mesenchymal stem cells loaded with paclitaxel inhibit tumor growth and angiogenesis
Authors: Alessandri Giulio , Bonomi Arianna , Coccè Valentina , Cavicchini Loredana , Invernici Gloria , Cristini Silvia , Sisto Francesca , Ferrari Maura , Ciusani Emilio , Cappelletti Graziella , Cartelli Daniele , Parati Eugenio , Pessina Augusto
Published: June 2012

Background: Mesenchymal stem cells (MSCs) may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that simple exposure of bone marrow (BM) derived MSCs to high doses of Doxorubicin, led them to acquire anti-proliferative potential towards co-cultured haematopoitic stem cells (HSCs). We thus hypothesized whether MSCs, once primed in vitro with anti-cancer drugs and then localized near cancer cells, can inhibit proliferation. Methods: Paclitaxel (PTX) was used to prime culture of mouse and human MSCs. Incorporation of PTX into MSCs (MSCsPTX) was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by MSCsPTX was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of MSCsPTX, while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting MSCsPTX with cancer cells in mice.

Results: MSCs were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. Primed MSCsPTX acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, MSCsPTX co-injected in immunodeficient mice with DU145 and B16 cancer cells, significantly delayed tumor takes and reduced tumor growth.

Conclusions: These data demonstrate that, without any genetic manipulation, MSCs can uptake and subsequently slowly release PTX, becoming a potential new tool for cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4354. doi:10.1158/1538-7445.AM2011-4354
Abstract 4355: Metformin potentiates the efficacy of radiotherapy of tumors by eliminating cancer stem cells
Authors: Choi Bo-Hwa , Lee Hyemi , Park Moon-Taek , Oh Eun-Taex , Song Min-Jeong , Song Chang Won , Park Heon Joo
Published: June 2012

Metformin, a widely used drug for type II diabetes, possess significant anti-cancer property. In a recent study, metformin was able to selectively kill chemoresistant cancer stem cells (CSCs). It has been known that CSCs are resistant to irradiation and, thus, they are responsible for the recurrence of cancer after radiotherapy. The purpose of the present study was to investigate whether metformin enhances the response of tumors to radiotherapy by eradicating radioresistant CSCs. An incubation with metformin at 0.1 mM – 10 mM for 72 hours caused clonogenic cell death in a dose-dependent manner in MCF-7 human breast cancer cells, MIA PaCa-2 human pancreatic cancer cells, and FSaII mouse fibrosarcoma cells. In addition, the clonogenic cell death was enhanced by using the combination of metformin and irradiation. We then investigated the effect of metformin on CSCs of the three cell lines, which were identified by ALDH-1 activity, CD24low/CD44+ expression, and SP method. Metformin was preferentially cytotoxic to CSCs in all these cell lines. The formation of mammospheres from MCF-7 cells was also markedly inhibited by metformin. Both metformin and irradiation activated AMPK and suppressed the mTOR-S6K signaling pathway. Combined treatment of cells with metformin and irradiation was far more effective in activating AMPK and suppressing the mTOR-S6K pathway. The combined effects of metformin and X-irradiation on the growth of FSaII tumors induced in the hind legs of C3H mice were studied. Metformin treatment at 50 – 100 mg/kg/day, i.p., slightly suppressed the tumor growth, but it strikely potentiated the response of tumors to 20 Gy X-irradiation in a single exposure. It was likely that metformin treatment depleted the radioresistant CSCs in the tumors, thereby markedly enhancing the response of tumors to irradiation. However, in addition to killing CSCs, the possibility that metformin radiosensitized CSCs as well as non-CSCs could not be excluded. Our results demonstrated for the first time that metformin is potentially useful for enhancing the efficacy of radiotherapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2011-4355
Abstract 4356: Combination use of anti-CD133 antibody and SSA lectin can effectively enrich cells with high tumorigenicity
Authors: Okudo Kumiko , Moriwaki Kenta , Haraguchi Naotsugu , Takeishi Shunsaku , Tanemura Masahiro , Mori Masaki , Miyoshi Eiji
Published: June 2012

A growing body of evidence has suggested that tumors are frequently composed of heterogeneous cell types, and that tumor initiation and growth are driven by a small subset of cells, termed cancer stem cells (CSCs) or tumor-initiating cells. CSCs can self-renew and also give rise to more differentiated progeny that comprise the bulk of a tumor. Glycans exhibit characteristic changes in their structures during development and thus have been used as markers for stem/progenitor cells. However, the glycan structures unique to CSCs have remained unknown. In this study, we examined glycan structures in CD133+CD13+ CSCs, which were recently found to have a high CSC ability, by means of lectin microarray. Seven sialylated glycan-recognizing lectins, MAL-I, SNA, SSA, TJA-I, ACG, ABA, and MAH, showed higher affinity to CD133+CD13+ CSCs than CD133+ cells with a lower CSC ability. In addition, we demonstrated that CD133+SSA+ cells isolated from Huh7 cells had a significantly higher ability to form tumors in NOD/SCID mice and spheres under serum-free conditions than CD133+SSA- cells. These results suggest that hepatic CSCs highly express sialylated glycans and that SSA lectin can be used as a tool for isolating CSCs. This study is the first report to demonstrate the characteristic glycan structures in CSCs and to indicate a new methodology involving lectins for isolating CSCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4356. doi:10.1158/1538-7445.AM2011-4356
Abstract 4360: Distinct energy metabolism in human pluripotent stem cells and differentiated cells regulated by UCP2
Authors: Zhang JIin , Khvorostov Ivan , Hong Jason , Oktay Yavuz , Vergnes Laurent , Nuebel Esther , Do Anna , Jung Hea-Jin , McCaffery Michael , Reue Karen , Koehler Carla , Teitell Michael
Published: June 2012

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbor a fused, branching mitochondrial network, with oxidative phosphorylation (OXPHOS) as the main energy source. A role for mitochondria in hPSC bioenergetics therefore remains uncertain. Here we show that hPSCs have functional respiratory complexes that consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis. To help maintain hPSC mitochondrial membrane potential, cell proliferation, and viability, glycolytic ATP is hydrolyzed by the mitochondrial F1F0ATP synthase. Uncoupling protein 2 (UCP2) regulates this unique pattern of hPSC energy metabolism while suppressing reactive oxygen species. Upon differentiation, UCP2 is repressed and cells shift from glycolysis and fatty acid oxidation to glucose oxidation dependency. Ectopic UCP2 expression during differentiation blocks this metabolic transition and blocks proper embryoid body formation. Combined, our data show that hPSC have distinct bioenergetics regulated by UCP2 and repression of UCP2 is required for metabolic reprogramming along differentiation

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2011-4360
Abstract 4361: The enzymatic activity of aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance
Authors: Moreb Jan S. , Ucar Deniz , Han Shuhong , Ostmark Blanca , Chang Lung-Ji
Published: June 2012

With the cancer stem cell theory, there has been an invigorated new interest in using ALDH activity as one marker for stem cells and such use became popular since a flow cytometry based assay, aldeflour staining, has become available. Diethylaminobenzaldehyde (DEAB), used in the aldeflour assay, has been published many years ago as a specific inhibitor for ALDH1 isoform. In this study, we explore the effects of specific ALDH isozymes, ALDH1A2 and ALDH2, and the specificity of DEAB as an inhibitor of their activity. We prepared lentiviral vectors containing the full length of either ALDH2 or ALDH1A2 cDNA and used them to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by spectrophotometric ALDH activity assay, Western blot analysis, RT-PCR, as well as aldeflour staining assay. In addition, gene expression profiles were assessed by quantitative real-time RT-PCR using micro fluidic cards (Taqman Low Density Arrays, TLDA, Applied Biosystem), covering 19 ALDH and three housekeeping genes. The expression levels were compared between sorted K562 with high versus low ALDH activity. The results show that we created 4 cell lines with successful high expression of each of the two enzymes and high ALDH activity which was detected by aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-HC and doxorubicin. In order to study the specificity of two known ALDH activity inhibitors, DEAB and tetraethylthiuram disulfide (disulfiram), we incubated each cell line with either inhibitor at 25 μM for 48 hr and then measured the remaining ALDH enzymatic activity. Our results show that both inhibitors reduced ALDH activity of both overexpressed isozymes, ALDH2 and ALDH1A2, by 70-80%. TLDA analysis revealed that K562 cells express high levels of isozymes in the ALDH1, ALDH3, ALDH7 and ALDH8 families. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that aldefluor assay is not specific assay for ALDH1A1 either. In addition, other ALDH isozymes seem to play a major role in the biology and drug resistance of various malignant cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4361. doi:10.1158/1538-7445.AM2011-4361
Abstract 4362: RBM3 overexpression promotes cancer stem cell expansion through activation of Notch-1 signaling
Authors: Ramalingam Satish , Subramaniam Dharmalingam , Jensen Roy A. , Anant Shrikant
Published: June 2012

Cancers are heterogeneous population of cells, a minor portion of which exhibit stem-like characteristics. Recent evidence suggests that these Cancer Stem Cells (CSCs) are responsible for tumor behavior. Recently, we reported that RNA binding protein RBM3 is overexpressed in various tumors of epithelial origin and downregulating its expression results in inhibition of tumor growth. Moreover, RBM3 affects the proliferation of CD34+ hematopoietic stem cell. Overexpression of RBM3 induces NIH-3T3 cells to transform and grow in an anchorage independent manner, and form tumors in athymic nude mice suggesting RBM3 is a proto-oncogene. Given these observations, we sought to determine whether RBM3 expression affects CSCs in solid tumors and its function in tumor initiation and/or metastasis. We have recently demonstrated that Doublecortin like Kinase-I (DCAMKL-1/DCLK1), a microtubule-associated kinase is a marker of quiescent intestinal stem cells. Immunohistochemical analyses of the NIH3T3-RBM3 tumors demonstrated an increase in the number of DCLK-1+ stem cells. Using fluorescence-activated cell sorting, we were able to isolate these DCLK-1+ stem cells from the bulk of the tumor and grow them in tissue culture dishes. We have named these cells as RNA binding protein-Induced Cancer Stem Cells (RICSCs). We next determined the stemness of the DCLK-1+ RICSCs. For this, we performed serial dilutions of the RICSCs starting at 10,000 cells and injected them into the flanks of nude mice. Even when 500 cells were injected, a primary tumor was observed at about 10 weeks; the histological analysis of the tissues revealing metastatic lesions in the lung, liver, bone and spleen. Recent studies with mammary and neuroectodermal tumors have suggested that inappropriate Notch activation promotes self-renewal and transformation of progenitor cells. We observed that there was a significant upregulation of activated Notch-1 expression in the NIH-3T3-RBM3 RICSCs. Inhibition of Notch-1 with a specific inhibitor, DAPT resulted in the arrest of RICSC-mediated tumor growth in nude mice. These data demonstrate that Notch signaling is essential for self-renewal in stem cells thereby affecting tumor growth. Taken together, these data imply that overexpression of RBM3 results in increased dedifferentiation into cancer stem cells that have an aggressive metastatic phenotype. These CSCs have great potentials for aiding in our understanding of oncogenesis and metastatic cascades.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4362. doi:10.1158/1538-7445.AM2011-4362