bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5336: Macrophage migration inhibitory factor (MIF) plays roles in proliferation, survival and migration of Ewing tumor cells through the activation of several tyrosine kinases
Authors: Kang Hyung Gyoo , Liu Xian Fang , Sorensen Poul HB , Triche Timothy J.
Published: June 2012

In normal cells, expression of cytokines and their mediated cell signaling pathways are tightly and accurately regulated. But in many tumors, it has been shown that cytokine signaling pathways are deregulated. Thus, identification and understanding of deregulated cytokines and their related pathways can provide the unique and promising opportunity for better-targeted therapies of cancer. MIF has been known to be a proinflammatory cytokine but recently it was shown that MIF plays roles in proliferation, survival and apoptosis in several tumors. In order to study the effects and molecular mechanisms of MIF on Ewing tumor cell growth and survival we established MIF deficient Ewing tumor cell lines using shRNA method. In our study it was found that the decreased MIF expression is significantly correlated to morphological change, deregulation of differentiation, decrease of proliferation and increase of apoptosis in Ewing tumor cells. Then several signal pathways were investigated to elucidate the molecular mechanism of MIF functions in Ewing tumor cells. It has been found that MIF is involved in the activation of Jak/Stat and Src pathways that regulate activation of Akt and Erk, which play key roles in the cell survival and proliferation. Related to the morphological change and mobility change in MIF-deficient cells it was found that Rac1 activation is highly regulated by MIF in Ewing tumors. In addition, it was found that activation of several kinases is closely related to the expression of MIF in Ewing tumors. Therefore our data implies that MIF might functionally contribute to Ewing tumor cell growth, survival, proliferation and metastasis, which suggests that MIF might be a promising target of Ewing tumor cells 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 5336. doi:10.1158/1538-7445.AM2011-5336
Abstract 5341: Combinatorial regulation of E2F target genes by the oncogenic ETS transcription factor EWS-FLI1
Authors: Schwentner Raphaela , Kauer Maximilian , Bilke Sven , Jug Gunhild , Meltzer Paul S. , Kovar Heinrich
Published: June 2012

Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer of the bone characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly binds 50% of E2F target genes and possibly cooperates with E2F transcription factors. Furthermore, several E2F factors were themselves found to be direct targets of EWS-FLI1. To unravel the hierarchical structure of this EWS-FLI1/E2F transcriptional network module we performed luciferase reporter gene assays in combination with DNA motif mutation analyses and ChIP-PCR.

The promoter regions of ten candidates for direct target genes of EWS-FLI1 and E2F factors were chosen for in-depth analysis. Corroborating the transcriptomic data, promoter activity of all ten genes showed reduced levels upon knockdown of EWS-FLI1.

By chromatin immunoprecipitation, we confirmed that EWS-FLI1 directly binds to the promoter regions. For some of those genes, mutation of only a single ETS binding site leads to a reduction of luciferase activity comparable to knockdown of EWS-FLI1 (for example the E2F promoters).

For the other selected EWS-FLI1 target candidates, mutation analysis and ChIP-PCR revealed more complex regulatory patterns.

In one example, simultaneous mutation of two ETS binding sites within the region directly bound by EWS-FLI1 resulted in a significant reduction of transcriptional activity. However, mutation of the single E2F binding site rendered the promoter completely insensitive to EWS-FLI1 regulation. Furthermore, ChIP-PCR revealed that upon EWS-FLI1 repression an EWS-FLI1 induced activating E2F factor is exchanged for a repressing E2F factor on the promoter of this gene. Our results imply that EWS-FLI1 uses several modes of interaction with E2F factors to control shared target genes.

This study was supported by grant 22328-B09 from the Austrian Science Fund FWF.

R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences.

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 5341. doi:10.1158/1538-7445.AM2011-5341
Abstract 5343: Aberrant activation of hedgehog signaling confers a poor prognosis in embryonal and fusion gene negative alveolar rhabdomyosarcoma
Authors: Zibat Arne , Missiaglia Edoardo , Rosenberger Albert , Pritchard-Jones Kathy , Shipley Janet , Hahn Heidi , Fulda Simone
Published: June 2012

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). The hedgehog (Hh) pathway has recently been implied in tumor formation and progression of various cancers. In the current study we investigated whether Hh pathway activation presents a general feature also of RMS and whether it bears prognostic impact. Here, we report that marker genes of active Hh signaling, i.e. Patched1 (Ptch1), Gli1, Gli3 and Myf5, are elevated in two distinct cohorts of RMS patients with a total number of 235 primary samples. Interestingly, all these marker genes of active Hh signaling were expressed at significantly higher levels in specific subtypes of RMS, i.e. ERMS and fusion gene negative ARMS, compared to fusion gene positive ARMS. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene negative ARMS, but not in fusion gene positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation in primary RMS samples. In support of this notion, the addition of Shh inhibits muscle differentiation in parallel experiments in muscle satellite cells and myoblasts. Moreover, we identify Myf5 is as a novel excellent class predictor for RMS by receiver operating characteristic (ROC) analysis. Most importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene negative RMS (P< 0.004) underscoring the clinical relevance of these findings. By identifying aberrant Hh activation in specific subgroups of RMS, our study has important implications for the development of molecular targeted therapies, such as small molecule Hh inhibitors, in RMS.

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 5343. doi:10.1158/1538-7445.AM2011-5343
Abstract 5348: In vivo and in vitro alveolar rhabdomyosarcoma cell growth is affected by TNK2 expression
Authors: Arnaldez Fernanda I. , Yeung Choh L. , Helman Lee J.
Published: June 2012

Rhabdomyosarcoma is the most common pediatric soft tissue tumor. The two major histologic subtypes are embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS); which have different genetic alterations: 11p15 LOH in ERMS and PAX3 (or PAX7)-FOXO1 translocation in ARMS. Despite utilization of aggressive multimodal therapy, patients with recurrent or metastatic disease remain essentially incurable. This is particularly true in patients with alveolar rhabdomyosarcoma. New therapies are needed to improve outcomes in this disease.

To detect genes that are critical for rhabdomyosarcoma cell growth and survival, we performed a loss-of-function shRNA screen in ARMS and ERMS cell lines using a regulated shRNA library that was delivered to RMS cells using retrovirus. We hypothesized that genes whose loss of function significantly influence the growth and survival of ARMS cells with relatively no impact on ERMS cells represent a critical vulnerability or “Achilles’ heel” for ARMS. We sorted our gene list using this approach. In this study, we identified TNK2 (tyrosine kinase, non-receptor 2; also known as ACK1) as a critical gene is ARMS cell survival and as a potential therapeutic target.

TNK2 is a non-receptor tyrosine kinase that integrates signals from different membrane receptors, including integrins, EGFR and IGFR. Reported downstream targets include CDC42 and AKT. The TNK2 gene is located at 3q29. TNK2 amplification has been described in prostate, lung and breast cancer and its role in invasion and metastasis has recently been suggested. However, its role in rhabdomyosarcoma has not been previously explored. We initially confirmed that TNK2 is uniformly expressed RMS. In addition, analysis of TNK2 expression in primary tumor samples of RMS using a publicly available database indicated that rhabdomyosarcomas with higher TNK2 expression show lower overall survival.

We used a tetracycline-inducible shRNA construct to knock down TNK2 expression in rhabdomyosarcoma cells. TNK2 knock down resulted in significant decrease in cell growth in ARMS. We characterized the specificity of our findings using 5 different shRNA sequences targeting this gene delivered via lentivirus. In addition, we have been able to rescue the effect of an shRNA targeting the 3’UTR of this gene using TNK2 cDNA. We validated our in vitro data in an in vivo model using tumor xenografts. Downstream pathways under investigation include but are not limited to AKT, MAPK and CDC42.

These data suggest that TNK2 could represent a therapeutic target in ARMS and highlight the role of loss of function shRNA screens to improve the understanding of tumor biology.

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 5348. doi:10.1158/1538-7445.AM2011-5348
Abstract 5352: Novel treatment approaches in retinoblastoma: Impact of combination therapy on tumor burden
Authors: Houston Samuel K. , Decatur Christina , Murray Timothy G. , Shah Nikesh , Cavalcante Ludimila , Pina Yolanda
Published: June 2012

Purpose: The purpose of the current study is to examine vessel targeting, chemotherapy, and mammalian target of rapamycin (mTOR) inhibitor agents in LHBETATAG retinal tumors and their impact on tumor burden.

Methods: Group A: Ten-week-old, LHBETATAG mice (n=30) received a single subconjunctival injection of anecortave acetate (AA; 1200, 600, 300, and 150 µg) delivered to right eyes only. Group B: Ten-week-old, LHBETATAG mice (n=30) received a single subconjunctival injection of AA (600, 300, and 150 µg) delivered to right eyes only, either during a cycle of carboplatin (six subconjunctival deliveries) or after the completed cycle. Carboplatin was delivered at the subtherapeutic concentration of 62.5 µg. All animals were euthanatized at 16 weeks of age, and the eyes were examined histopathologically. Group C: Eighteen-week-old, LHBETATAG mice received (n=30) subconjunctival injections of rapamycin once weekly for two consecutive weeks (0.00333, 0.167, 3.33, and 6.67 mg/kg). Tumor sections were analyzed for tumor burden with immunohistochemistry techniques.

Results: A statistically significant reduction in tumor burden was detected after a single periocular injection of AA. The reduction of tumor burden followed a U-shaped dose-response curve. Tumor burden was significantly decreased when AA and carboplatin were combined. However, varying doses and delivery schedule of these agents had significant impact on the effectiveness of the combined treatment. The most effective scheme was delivering a low dose (150-300 µg) of AA after a complete cycle of carboplatin. Reduction in tumor burden were significantly different between rapamycin doses and control (p<0.002). Eyes treated with rapamycin at 0.167, 3.33 and 6.67 mg/kg showed a significant decrease in tumor burden in comparison with the vehicle control group (p=0.019, p=0.001, p=0.009, respectively) and the 0.00333 mg/kg dose response (p=0.023, p=0.001, p=0.010, respectively). Histopathological evaluation showed no signs of retinal toxicity to either treatment used.

Conclusions: AA, as monotherapy or as adjuvant therapy, significantly controlled tumor burden in a murine model of retinoblastoma. Moreover, adjuvant therapy enabled the use of typically subtherapeutic carboplatin doses without decreasing efficacy of the therapy. Inhibition of mTOR reduced tumor burden during late disease in the LHBETATAG retinoblastoma tumor model. Rapamycin may have a role in combination with chemotherapy or other adjuvant therapies to enhance retinoblastoma tumor control.

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 5352. doi:10.1158/1538-7445.AM2011-5352
Abstract 5357: Pediatric preclinical evaluation of the PARP inhibitor olaparib
Authors: Norris Robin E. , Adamson Peter C. , Fox Elizabeth
Published: June 2012

Purpose: Olaparib (AZD2281) is an oral poly(ADP)-ribose polymerase (PARP) inhibitor in clinical development as a single agent and in combination with cytotoxic therapy in adults with solid malignancies. In BRCA-1 deficient tumors, olaparib concentrations of 30 nM result in significant PARP inhibition (Rottenberg, PNAS, 2008). Adult patients appear to tolerate olaparib doses associated with a Cmax of 10 µM (Fong, NEJM, 2009). We evaluated the interaction of olaparib with commonly used cytotoxic drugs in a panel of pediatric solid tumor cell lines.

Methods: In vitro growth inhibition was assessed using the sulforhodamine B assay in pediatric solid tumor cell lines including: osteosarcoma (MG-63), neuroblastoma (NGP), Ewings sarcoma (RD-ES), medulloblastoma (Daoy), and rhabdomyosarcoma (RD). IC50's were determined following a 120 h exposure to vehicle, olaparib, cytotoxic agent (carboplatin, melphalan, irinotecan, doxorubicin, vincristine) or olaparib in combination with each cytotoxic agent. The combination index (CI) of Chou-Talalay was used to test for synergy of combinations.

Results: As a single agent, olaparib was cytotoxic in all cell lines studied, with a median (range) IC50 of 1.7 (0.3-5.3) μM. The Ewings sarcoma cell line was the most sensitive line with a mean (SD) IC50 of 0.7 (0.3) μM. The combination of olaparib with melphalan or irinotecan was synergistic (CI < 1), while the addition of olaparib to doxorubicin or carboplatin appeared additive to synergistic (CI < 1). The combination of olaparib and vincristine trended toward antagonism (CI > 1).

Conclusions: The addition of olaparib to DNA damaging agents resulted in additive or synergistic growth inhibition in a panel of pediatric solid tumor cell lines. Further preclinical and clinical evaluation to determine if olaparib can enhance the efficacy of current pediatric chemotherapy regimens is warranted.

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 5357. doi:10.1158/1538-7445.AM2011-5357
Abstract 5363: Dual targeting of mTORC1/C2 complexes enhances histone deacetylase inhibitor-mediated antitumor efficacy in primary HCC cancer in vitro and in vivo
Authors: Shao Huanjie , Tang Haikuo , Gao Chun , Zhang Hao , Dy Grace , Yu Chunrong , Adjei Alex , Roberts Lewis
Published: June 2012

The mammalian target of rapamycin (mTOR) consists of two distinct complexes, mTORC1 and mTORC2. mTOR plays a pivotal role in Hepatocellular Carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 only moderately enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity. This limited effect may be attributable to the increase of AKT activation through S6K1-IRS1-PI3K negative feedback loop triggered by selective mTORC1 inhibition. While as AKT is the downstream effector of mTORC2, we hypothesize that dual targeting of mTORC1/C2 would be more efficient in suppressing AKT and in enhancing anti-tumor activity of HDACi. In this study, the interactions between mTOR kinase inhibitors (mTORKis) and HDACis were examined in vitro using established HCC cell lines and in vivo using patient-derived HCC tumor xenografts on SCID mice. Our results demonstrate that inhibition of both mTORC1/2 not only effectively blocked mTORC1 signaling, but also abrogated negative-feedback activation of AKT caused by mTORC1 inhibition. Furthermore, inactivation of mTORC1/C2 by mTORKis substantially enhanced HDACi-induced anti-tumor activity. Either dysfunction of mTORC2 by knockdown of Rictor (a component of mTORC2), or diminution of AKT1 significantly lowered the threshold of HDACi-induced apoptosis. Lastly, in vivo study indicated that the combination of AZD8055 and Vorinostat yielded much greater tumor-growth inhibition compared to either agent alone in primary HCC xenografts without obvious adverse effects. Conclusion: Dual mTORC1/2 inhibition is more efficient than selective mTORC1 inhibition in enhancing anti-tumor activity of HDACi due to abrogation of negative-feedback activation of AKT induced by selective mTORC1 inhibition. Our findings suggest that combining regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC.

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 5363. doi:10.1158/1538-7445.AM2011-5363
Abstract 5369: Prostate cancer radiosensitization through PARP-1 hyperactivation
Authors: Dong Ying , Bey Erik A. , Li Longshan , Boothman David A.
Published: June 2012

The clinical experimental agent, beta-lapachone (Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether beta-lapachone may warrant clinical evaluation as a radiosensitizer. Beta-lapachone killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species (ROS), irreversible DNA single strand breaks (SSBs), PARP-1 hyperactivation, NAD+/ATP depletion, and µ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), beta-lapachone radiosensitized NQO1+ prostate cancer cells, under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSBs, gamma-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of µ-calpain-induced programmed cell death. Radiosensitization by beta-lapachone was blocked by the NQO1 inhibitor, dicoumarol, or the PARP-1 inhibitor, DPQ. In a mouse xenograft model of prostate cancer, beta-lapachone synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ∼60% of human prostate tumors evaluated relative to adjacent normal tissue, where beta-lapachone might be efficacious alone or in combination with radiation. Our findings offer a rationale for clinical assessment of beta-lapachone (Arq501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation. Since similar data have been found for all NQO1 endogenously over-expressing cancers, including nonsmall cell lung, pancreatic and breast cancer, this strategy should have a broad use for many cancers and in combination with many DNA damaging agents used for cancer therapy. This work was supported by NIH grant 2 R01 CA102792 to DAB

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 5369. doi:10.1158/1538-7445.AM2011-5369
Abstract 5376: Potentiation of the anti-proliferative and cytotoxic activity of ARQ 197 & gemcitabine with a pulsatile exposure regimen
Authors: Chen Chang-Rung , Abbadessa Giovanni , McSweeney Denise , Szwaya Jeffrey , Chan Thomas CK , France Dennis S.
Published: June 2012

ARQ 197 is a small molecule that inhibits c-MET kinase and is being evaluated clinically both as a monotherapy and in combination with other anti-cancer agents, including gemcitabine. Prompted by clinical observations, in vitro studies were conducted to explore the combination of ARQ 197 and gemcitabine with the goal of determining the most effective exposure regimen and to further refine the strategy of an ongoing phase 1b combination clinical trial. Gemcitabine and ARQ 197 both induce cell cycle arrest in cancer cells, but in different phases. Gemcitabine induces a G1/S-phase arrest, while ARQ 197 arrests cells in the G2/M phase. We hypothesized that the efficacy of combining these two agents might be enhanced by deliberate pulsatile treatment with ample time allowed for gemcitabine-treated cells to re-enter the cell cycle and to therefore potentially increase their vulnerability to ARQ 197-induced cell cycle arrest and apoptosis. A 7-day MTS cytotoxicity assay was employed in which the cells were seeded on Day 0, treated with gemcitabine for 24 h on Day 2, drug free medium added on Day 3, and ARQ 197 added on Day 5 for 24 h and then replaced with drug free medium. Cell viability was assayed with MTS reagent at the end of Day 7. To determine whether this pulsatile schedule was more advantageous then a regimen with no intervening “drug holiday”, control cultures were not replenished with drug free medium on Day 3 and ARQ 197 was added on Day 5. When the pulsatile schedule was employed, the combinatorial cytotoxicity of these two agents was significantly enhanced in several cancer cell lines (including NSCLC, bladder, pancreatic, breast, ovarian and uterine carcinomas). Mechanistic studies confirmed the G1/S-phase arrest of gemcitabine-treated cells, and G2/M arrest was observed following exposure to ARQ 197 in this regimen. Furthermore, this potentiation was also observed if ARQ 197 was exposed to the cancer cells as the first agent on Day 2 and gemcitabine added as the second agent on Day 5, as long as the pulsatile regimen was employed. These data suggest that modifications to dosing schedules of ARQ 197 and gemcitabine may improve the potential for synergistic anti-tumor activity in the clinic.

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 5376. doi:10.1158/1538-7445.AM2011-5376
Abstract 5378: SB225002 induces apoptosis in acute lymphoid leukemia cells
Authors: Vasconcellos Jaira F. , Laranjeira Angelo B. A. , Leal Paulo C. , Nunes Ricardo J. , Yunes Rosendo A. , Brandalise Silvia R. , Libermann Towia A. , Zerbini Luiz F. , Yunes José A.
Published: June 2012

Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. We have recently shown that the leukemic bone marrow (BM) contains high levels of interleukin-8 (IL-8), whose cognate receptor CXCR2 is expressed by most primary ALL cells. ALL cell lines are sensitive to SB225002 (N-(2-hydroxy-4-nitrophenyl)-N’-(2-bromophenyl)urea), a CXCR2 antagonist. Surprisingly, the inhibitory effect of this drug was found to be independent of CXCR2 expression by leukemic cells. The current study was conducted to determine the mechanism of action of SB225002 against ALL cells, using as prototype the T-ALL cell line Jurkat. Cell cycle analysis showed that SB225002 induces G2-M cell cycle arrest. Transcriptional profiling by microarray analysis revealed that SB225002 induces 174 genes and represses 41 genes. SB225002 induced-apoptosis triggered a transcriptional program of genes involved in activation of the JUN and p53 pathways and inhibition of the TNFα pathway. Results were further confirmed by Q-PCR and/or Western blot analysis. Treatment of Jurkat cells with SB225002 also induced the activation of phospho-p44/42 MAPK (ERK1/2), which was suspected to be an anti-apoptotic pathway triggered by injured cells. In fact, the inhibition of ERK1/2 with pharmacologic inhibitors, in combination with SB225002, resulted in the enhancement of apoptosis, suggesting the blockage of ERK1/2 pathway as a strategy to increase the sensitivity of ALL cells toward SB225002. Connectivity Map analysis of the SB225002 gene signature demonstrates a positive correlation with the signatures for PI3K and HSP90 inhibitors (connectivity score 1 and 0.944, respectively), which mediate apoptosis through effects on AKT, TNFα and NF-κB, while SB225002's mediated-apoptosis demonstrated only to inhibit TNFα pathway. Furthermore, SB225002 gene signature demonstrates a positive correlation with Nocodazole and Podophyllotoxin (connectivity score 0.895 and 0.859, respectively), which are inducers of cancer cell apoptosis by interfering with microtubule polymerization and inhibiting topoisomerase II, respectively. Our data demonstrate for the first time that JUN, p53, and TNFα pathways are modulated by SB225002, and that ALL cells are sensitized to SB225002-induced apoptosis after phospho-p44/42 MAPK inhibition. Overall these data support SB225002's therapeutic potential in ALL.

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 5378. doi:10.1158/1538-7445.AM2011-5378
Abstract 5390: A serotype 5/3 adenovirus expressing MDA-7/IL-24 infects renal carcinoma cells and promotes toxicity of agents that increase ROS and ceramide levels
Authors: Bareford Mary Danielle , Hamed Hossein A. , Park Margaret , Grant Steven , Fisher Paul , Dent Paul
Published: June 2012

It recognized that agents that generate reactive oxygen species (ROS) enhance MDA-7/IL-24 lethality. The present studies focused on clarifying how such agents enhanced MDA-7/IL-24 toxicity in renal cell carcinoma cells (RCCs). Infection of RCCs with a tropism-modified serotype 5/3 adenovirus expressing MDA-7/IL-24, Ad.5/3-mda-7, caused plasma membrane clustering of CD95 and CD95 association with pro-caspase 8, effects that were enhanced by combined exposure to 17AAG, As2O3 or 4HPR, and which correlated with enhanced cell killing. Knock down of CD95 or expression of c-FLIP-s blocked enhanced killing. Inhibition of ROS generation, elevated cytosolic Ca2+, or de novo ceramide synthesis blocked Ad.5/3-mda-7 +/- agent-induced CD95 activation and the enhancement of apoptosis.Ad.5/3-mda-7-increased ceramide levels in a PERK-dependent fashion that were responsible for elevated cytosolic Ca2+ levels that promoted ROS generation; 17AAG did not further enhance cytokine-induced ceramide generation. In vivo, infection of RCC tumors with Ad.5/3-mda-7 suppressed the growth of infected tumors that was enhanced by exposure to 17AAG. Our data indicates that in RCCs Ad.5/3-mda-7-induced ceramide generation plays a central role in tumor cell killing and inhibition of multiple signaling pathways may have utility in promoting MDA-7/IL-24 lethality in renal cancer.

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 5390. doi:10.1158/1538-7445.AM2011-5390
Abstract 5394: Dual inhibition of the androgen receptor by ligand blockade and antisense-mediated downregulation is associated with synergistic antitumor activity model of prostate cancer
Authors: Zhang Yixian , Dumble Melissa , Castaneda Stephen , Milesky Mary , Kim Steven , Qu Zhengxing , Greenberger Lee M. , Horak Ivan D.
Published: June 2012

Background: Although new agents to treat prostate cancer have recently been approved, and new investigational drugs (Abiraterone and MDV3100) that mediate hormone deprivation show promising data from late-stage clinical trials, these therapies typically extend overall survival up to 4 months in patients with advanced disease who have failed conventional androgen deprivation therapy. Hence, there continues to be great medical need. EZN-4176 is a novel LNA-based antisense oligonucleotide (EZN-4176) that down regulates androgen receptor (AR) expression. Previously, antitumor activity of EZN-4176 in multiple xenograft models including both androgen-sensitive and castrate-resistant tumors was shown. More importantly, a synergistic effect was also found when EZN-4176 was combined with MDV3100. Further exploration of the mechanistic underpinning of the effect was explored here. Methods: The mRNA, growth, luciferase activity, protein, and prostate specific antigen (PSA) were evaluated by qRT-PCR, MTT, SteadyGlo, western blot analysis, and ELISA assay, respectively. The effect of EZN-4176 on AR transcriptional activity was evaluated in cells stably express luciferase gene regulated by AR. In vivo, the effect of EZN-4176 on AR transcriptional activity was evaluated in a bone tumor model developed by intratibia injection of C4-2b-AR-luc cells. Results: Combination of EZN-4176 with MDV3100 resulted in a much improved inhibitory effect in colony formation assay in vitro. This effect was not associated with enhanced efficiency of AR mRNA down-modulation as treatment with EZN-4176 with or without MDV3100 showed similar effects on the level of AR mRNA. However, when AR transcriptional activity was examined, we found that the combined effect was greater than each agent alone. In C4-2b-AR-luc bone tumor model, EZN-4176 potently and specifically down-modulated AR-luciferase reporter activity. Further examinations of the anti-tumor effect in this model will help understand the potential of the combinatorial effect in a highly relevant disease model. Conclusions: Our preliminary data suggest that the synergistic anti-tumor effect of EZN-4176 in combination with MDV3100 is in part associated with much reduced transcriptional activity of AR. EZN-4176 alone or in combination with anti-androgens offers great potential in treating patients with advanced prostate cancer.

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 5394. doi:10.1158/1538-7445.AM2011-5394
Abstract 5395: Mutant oncolytic Herpes simplex virus expressing yeast cytosine deaminase in combination with 5-FC: Viral replication and prodrug bioactivation kinetics in vivo
Authors: Yamada Suguru , Kuroda Toshihiko , Fuchs Bryan C. , He Xiaoying , Supko Jeffrey , McGinn Christopher M. , Schmitt Anthony , Lanuti Michael , Tanabe Kenneth K.
Published: June 2012

Background: Herpes simplex virus 1 (HSV-1) expressing yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). However, a greater understanding of the interactions between the cellular response to 5-FC activation and viral replication is required to establish optimal dosing schedules for virus and prodrug administration.

Methods: Using the flip flop BAC system we developed a HSV-1 mutant expressing the yCD gene in the ICP6 locus in the HSV strain Patton, termed bMP6-CMVFcumB. BALB/c mice with MC26 flank tumors were treated by intratumoral injection of bMP6-CMVFcumB and 5-FC was administered intraperitoneally at different initiation times for 6 days. We also investigated different durations of 5-FC administration after initiation on Day 3 after viral injection. To measure viral replication and transgene expression, MC26 tumors on flanks were harvested at different time points between Day 0 and Day 21 following virus injection. Tumor lysates were analyzed by quantitative real-time PCR to assess genomic HSV-DNA, viral plaque assay to assess replicative virions and LacZ staining to assess transgene expression. 5-FU levels in serum and tumor were measured by HPLC at specific time points following administration of 5-FC on Day 3 after viral injection.

Results: Intratumoral viral genomic DNA copy number and viral titers peaked on Day 3 after viral injection and gradually decreased afterwards. And consistent with this, 5-FC administration on Day 3 post viral injection was more effective than administration on Day 6. Virus was detected for as long as 2 weeks after viral injection. And consistent with this finding, administration of 5-FC for 12 days was superior to 6 days, and administration for 18 days provided no added benefit over 12 days. The peak level of 5-FU in tumors was seen on Day 6 after viral injection and this value decreased gradually afterwards. Analysis of serum samples demonstrated that 5-FU was present at very low levels on Day 6, but it could not be detected at any other time.

Conclusion: Viral replication and transgene expression can be effectively monitored over time and correlated with therapeutic response. Our results suggest that the most potent effect by this combination therapy is observed when 5-FC is initiated at the onset of peak viral and transgene expression and continued for the duration of 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 5395. doi:10.1158/1538-7445.AM2011-5395
Abstract 5403: Pro-apoptotic properties of Bag-1 peptides in the control of prostate cancer cell growth
Authors: Maddalo Danilo
Published: June 2012

Purpose of the study: The purpose of the study is to identify sequence(s) in Bag-1 proteins responsible for growth inhibition and to eventually derive a short peptide for cancer therapy.

Introduction: Bag-1 family of proteins consists of four members (Bag- 1L, 1M, 1 and 1S) originating from different translation-initiation sites on the same mRNA. Clinical reports have shown a dual role of the Bag-1 proteins in tumor progression and in the inhibition of tumor growth. In this work we have identified a 19 amino acid sequence in the Bag-1 proteins that reduces prostate cancer cell growth ex vivo and in vivo.

Experimental procedures: Inhibition of prostate cancer cell growth was analyzed in clonogenic assay and in athymic nude mouse xenograft models. Histological analysis for apoptosis was carried out on paraffin-embedded tumor samples. Structural analyses were carried out by NMR and circular dichroism. Interaction partners of the Bag-1 peptides were analyzed by GST-pull down and by co-immunoprecipitation assays. Significance of cellular levels of glucose-regulated proteins (GRP) 75 and 78 for the growth inhibitory action of the peptide was demonstrated by RNA interference experiments.

Results: Overexpression of Bag-1 proteins in 22Rv.1 prostate tumor cells reduced the growth of these cells in a colony forming assay. A study making use of several deletion mutants identified a 69 amino acid Bag-1 sequence as responsible for the growth inhibition. NMR and circular dichroism studies showed this peptide to be unstructured but it nonetheless interacted with the substrate binding domain of GRP 75 and 78. The cellular levels of the GRPs were shown by RNA interference studies to be essential for the action of the peptide. Overexpression of the peptide in the 22Rv1 cells decreased growth in proliferation assay and in xenograft tumor models. The decreased growth in vivo was demonstrated to be due to apoptosis of the Bag-1 peptide expressing cells as showed by immuohistochemical analysis. The growth inhibitory activity could be narrowed down to a 7 amino acid core within a 19 amino acid Bag-1 peptide sequence. Amino acid exchanges in the core region impaired binding of the peptide to the GRPs and inhibition of tumor cell growth. Colony formation assay using a wide panel of prostate cells showed that the 19 amino acid peptide inhibits growth of only malignant but not benign cells and this correlates with the level of expression of the two GRP target proteins in the cells.

Conclusion: Together these results demonstrate that a short peptide sequence derived from Bag-1 proteins that binds GRP75/78 specifically inhibits the growth of prostate cancer 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 5403. doi:10.1158/1538-7445.AM2011-5403
Abstract 5405: Apoptosis induction in cancer cells by the simultaneous transduction of p53 and artificial miRNAs based on the screening of genome-wide shRNA library
Authors: Idogawa Masashi , Sasaki Yasushi , Kashima Lisa , Shinomura Yasuhisa , Imai Kohzoh , Tokino Takashi
Published: June 2012

Gene transfer involving p53 is viewed as a potentially effective cancer therapy, but does not result in a good therapeutic response in all human cancers. Several p53 target genes have an inhibitory effect on p53-mediated apoptosis. Therefore, if the induction of these genes by p53 is selectively suppressed, the therapeutic effectiveness would be improved.

In previous study, we constructed a replication-deficient recombinant adenovirus that encoded co-cistronic p53 and artificial microRNAs (miRNAs) targeting p21, a representative p53 target gene which have an anti-apoptotic effect, resulting in p53 expression and the suppression of p21 induction simultaneously. In colorectal and hepatocellular carcinoma cells, the infection with the adenovirus vector augmented apoptosis and suppressed tumor growth as compared to an adenovirus that expressed p53 alone in vitro and in vivo.

In this study, we simultaneously infected the vectors which express artificial miRNAs targeting other anti-apoptotic genes and evaluated the therapeutic effectiveness of p53 expression in combination with the suppression of several anti-apoptotic p53 target genes. Co-infection with these vectors suppressed the induction of anti-apoptotic target genes and induced apoptosis more efficiently in cancer cells.

To identify other RNAi targets which prevent p53-induced apoptosis, we have screened the genome-wide lentiviral shRNA library in the cancer cells which resist p53-induced apoptosis. After the infection with p53 or control adenovirus, the cell populations expressing each shRNA were quantified by microarray. As a result, we found that several cell populations expressing specific shRNAs were significantly decreased in p53-infected cells compared with control cells. This result indicated that the suppression of specific targets by RNAi restored the apoptotic response by p53 transduction in cancer cells which originally resist p53-induced apoptosis.

These results suggest that the transduction of p53 and specific miRNAs may be effective as a 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 5405. doi:10.1158/1538-7445.AM2011-5405
Abstract 541: The oral proteasome inhibitor ONX 0912 has bone anabolic effects in addition to anti-myeloma effects
Authors: Hurchla Michelle A. , Hornick Mary C. , Li Aixiao , Collins Lynne , Kirk Christopher J. , Piwnica-Worms David , Vij Ravi , Tomasson Michael H. , Weilbaecher Katherine N.
Published: June 2012

Multiple Myeloma (MM) is a cancer of antibody producing plasma cells in which tumors largely reside in and destroy the bone microenvironment. Drugs that inhibit proteasomal degradation of intracellular proteins such as those crucial for cell growth, have demonstrated significant efficacy in the treatment of MM. ONX 0912, is a next generation proteasome inhibitor that acts irreversibly and has the significant advantage of oral delivery. Pharmacokinetic data suggest that the in vivo serum half life of ONX 0912 is approximately 4 hours. We found that a physiologic 4 hour pulse treatment with low nanomolar doses of ONX 0912 effectively decreased the viability of human MM cell lines through the inhibition of proliferation and induction of apoptosis with similar efficacy as continuous drug administration in vitro. Because MM induces significant bone loss through the tumoral production of factors involved in the recruitment of bone resorping osteoclasts (OC) and suppression of bone forming osteoblasts (OB), we also evaluated the effects of ONX 0912 on bone cell viability and function. Bortezomib, an intravenous proteasome inhibitor in clinical use with a serum half-life of one hour, has been reported to suppress OC viability and enhance OB function. We found that culturing of primary murine macrophages, preosteoclasts, and OC under continuous incubation with bortezomib and ONX 0912 decreased cell viability, although at doses greater than 100-fold higher than those affecting MM cells. However, when primary macrophages and OC were incubated with a physiologic 1 hour pulse of bortezomib or 4 hour pulse of ONX0912, these cells were resistant to killing with in vitro osteoclastogenesis being modestly inhibited. In contrast, physiologic 4 hour pulse exposure of ONX 0912 to primary OB cultures, resulted in enhanced in vitro differentiation and mineralization. Likewise, oral administration of ONX 0912 to non-tumor bearing C57BL/6 mice significantly increased trabecular bone volume and bone mineral density. We then evaluated the effect of ONX 0912 on MM tumor burden and tumor associated bone loss in vivo. Oral administration of ONX 0912 significantly reduced the growth of human RPMI8226 MM cells in long bones of NOD-SCID-IL-2Rγc-/- mice and protected mice from bone loss compared to vehicle treated mice. These data suggest that, in addition to its anti-tumor effects, ONX 0912 may also shift the bone microenvironment during MM from a catabolic state to an anabolic state, reducing osteolysis, promoting bone formation and decreasing skeletal complications of MM.

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 541. doi:10.1158/1538-7445.AM2011-541
Abstract 5411: Development of SCLC specific nanoparticle-mediated therapeutic gene delivery
Authors: Kamat Chandrashekhar D. , Shmueli Ron B. , Sunshine Joel C. , Connis Nick , Green Jordan J. , Hann Christine L.
Published: June 2012

Lung cancer is the most common cause of cancer-related deaths worldwide. Small cell lung cancer (SCLC) is a particularly aggressive form of lung cancer characterized by early metastatic behavior. Even with currently recommended therapy, the average survival of SCLC patients is less than 1 year. There is an urgent need for novel treatments. The majority (70-90%) of SCLC cases possess alterations in p53 and Rb. This relative genetic homogeneity makes SCLC an attractive context in which to develop and test novel gene therapy approaches. We have initiated studies using non-viral, poly-β amino acid ester (PBAE) nanoparticle delivery of gene therapy in SCLC. PBAE polymers possess numerous advantages over viral delivery systems such as structural diversity, cost effectiveness, low immunogenicity and higher stability. PBAEs encapsulate DNA to form biodegradable cationic nanoparticles and have been studied in a variety of cancer cell types and in primary endothelial cells. This is the first study of PBAEs in SCLC.

A PBAE polymer library was screened in two SCLC lines using a luciferase-based high-throughput screen (HTS). Thirty-two of 112 polymers were further characterized in the H446 SCLC cell line using a CMV-GFP reporter construct and analyzed by qualitative (microscopy) and quantitative (flow cytometry) methods. This secondary screen revealed that the polymers had variable transfection efficiencies ranging from 8-55%. Four polymers, 446LG, 447LG, 456LG and 457 exhibited high transfection efficiencies (42-55%) in two SCLC cell lines, including one suspension line. Using PBAEs complexed with Cy5-labeled DNA, we were able to demonstrate that >90% cells bind and internalize the nanoparticles. We have initiated studies to better elucidate bottlenecks between nanoparticle uptake and gene expression. Additionally, preliminary studies in tumor-bearing athymic nude mice demonstrated gene uptake and expression via intratumoral injection of 457 and 446LG polymer/DNA complexes.

Ongoing studies include further optimization of PBAEs for in vitro gene expression and systemic in vivo delivery. In parallel, we are performing genetic characterization of patient-derived primary SCLC xenografts to verify relevant gene targets. Taken together, our data supports further development of a non-viral PBAE-based gene delivery approach in SCLC.

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 5411. doi:10.1158/1538-7445.AM2011-5411