bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 804: Disseminated tumor cell heterogeneity and dormancy in prostate cancer.
Authors: Lam Hung-Ming , Chéry Lisly , Coleman Ilsa , Lakely Bryce , Larson Sandy , Coleman Roger , Brown Lisha , Doan Kathy , Noteboom Jennifer , Zhang Xiaotun , True Lawrence , Nelson Peter , Montgomery Bruce , Lange Paul , Snyder Linda , Vessella Robert , Morrissey Colm
Published: August 2014

Prostate cancer (PCa) can remain in the bone marrow for a prolonged period of time (>5 years) while the patient shows no evidence of disease before the cancer eventually recurs. Dormant cancer cells can be detected in bone, the principal metastatic site of PCa, and these bone-homing cancer cells are known as disseminated tumor cells (DTC). Little information is available on the heterogeneity and dormancy of DTC in PCa. In this study, we isolated and compared the gene expression profile of individual DTC (n=45) from the bone marrow of 4 PCa patients with no evidence of disease and 5 patients with advanced disease. Using principle component analysis and cluster analysis of the 1000 most variable genes, we determined the heterogeneity of the DTC population within each patient. To identify a dormancy signature from DTC in the bone marrow and primary PCa cells, we carried out two gene expression analyses: DTC in patients with no evidence of disease vs. those with advanced disease, and primary PCa tissues from patients with a short vs. long dormancy period post radical prostatectomy (8-86 months). Genes associated in other cancers with cellular senescence, cell-cycle inhibition, and dormancy were analyzed. Candidate genes from both gene expression arrays were validated at the protein level by a tissue microarray consisting of 64 primary PCa cases that recurred after either a short or long dormancy period post radical prostatectomy (6-121 months).

The identification of heterogeneous gene signatures in DTC and novel proteins that promote dormancy will guide the development of possible biomarkers and therapeutic targets to prevent PCa recurrence, possibly by either eliminating DTC or inhibiting their escape from dormancy.

Citation Format: Hung-Ming Lam, Lisly Chéry, Ilsa Coleman, Bryce Lakely, Sandy Larson, Roger Coleman, Lisha Brown, Kathy Doan, Jennifer Noteboom, Xiaotun Zhang, Lawrence True, Peter Nelson, Bruce Montgomery, Paul Lange, Linda Snyder, Robert Vessella, Colm Morrissey. Disseminated tumor cell heterogeneity and dormancy in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2013-804
Abstract 805: Simultaneous copy number gains of NUPR1 and ERBB2 predicting poor prognosis in early-stage breast cancer.
Authors: Chung Yeun-Jun , Jung Seung-Hyun , Lee Ahwon , Yim Seon-Hee
Published: August 2014

The full extent of chromosomal alterations and their biological implications in early breast carcinogenesis has not been well examined. In this study, we aimed to identify chromosomal alterations associated with poor prognosis in early-stage breast cancers (EBC). A total of 145 EBCs (stage I and II) were examined in this study. We analyzed copy number alterations in a discovery set of 48 EBCs using oligoarray-comparative genomic hybridization. In addition, the recurrently altered regions (RARs) associated with poor prognosis were validated using an independent set of 97 EBCs. A total of 23 RARs were defined in the discovery set. Six were commonly detected in both stage I and II groups (> 50%), suggesting their connection with early breast tumorigenesis. There were gains on 1q21.2-q21.3, 8q24.13, 8q24.13-21, 8q24.3, and 8q24.3 and a loss on 8p23.1-p22. Among the 23 RARs, copy number gains on 16p11.2 (NUPR1) and 17q12 (ERBB2) showed a significant association with poor survival (P=0.0186 and P=0.0186, respectively). The patients simultaneously positive for both gains had a significantly worse prognosis (P=0.0001). In the independent replication, the patients who were double-positive for NUPR1-ERBB2 gains also had a significantly poorer prognosis on multivariate analysis (HR=7.31, 95% CI 2.65-20.15, P=0.0001). The simultaneous gain of NUPR1 and ERBB2 can be a significant predictor of poor prognosis in EBC. Our study will help to elucidate the molecular mechanisms underlying early-stage breast cancer tumorigenesis. This study also highlights the potential for using combinations of copy number alterations as prognosis predictors for EBC.

Citation Format: Yeun-Jun Chung, Seung-Hyun Jung, Ahwon Lee, Seon-Hee Yim. Simultaneous copy number gains of NUPR1 and ERBB2 predicting poor prognosis in early-stage breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 805. doi:10.1158/1538-7445.AM2013-805
Abstract 821: Subgroups of urothelial carcinoma of the bladder defined by genomic analysis.
Authors: Hurst Carolyn D. , Platt Fiona M. , Taylor Claire F. , Knowles Margaret A.
Published: August 2014

Urothelial carcinoma (UC) of the bladder is a heterogeneous disease and presents a significant challenge in disease management. The divergence in clinical behaviour of tumors of similar stage and grade indicates the presence of molecular subgroups. In this study we aimed to identify novel genomic subgroups of UC to provide improved subclassification at diagnosis. We assessed 160 bladder tumors comprising all stages and grades and including 49 high-stage T1 (T1G3) tumors for genome-wide copy number alterations and mutations in 8 key genes (AKT1, FGFR3, HRAS, KRAS, NRAS, PIK3CA, TP53, TSC1) implicated in UC. This revealed the existence of potential genomic subclasses of the “gold-standard” stage/grade groups of UC. The clinical management of patients with T1G3 tumors is particularly challenging as this subset of tumors exhibit highly divergent behaviour. Three major subgroups of T1G3 tumors were identified that differed with respect to copy number alterations and to FGFR3 and TP53 mutation status. One of these, which showed a prevalence of copy number losses, had the highest rate of stage progression/metastasis. We also identified novel regions of copy number alteration, concomitant molecular events, and relationships between molecular events and clinico-pathological features. Tumors harbouring FGFR3 mutations were found to be more chromosomally stable than their wild-type counterparts and FGFR3 mutation was mutually exclusive with overrepresentation of 8q in non-muscle-invasive tumors. In muscle-invasive (MI) tumors, losses of regions on 10q (including PTEN), 16q and 22q and gains on 10q, 11p, 12p, 19p and 19q were positively associated with metastasis. TP53 mutation in MI tumors was positively associated with losses on 16p, 2q, 4q, 11p, 10q, 13q, 14q, 16q and 19p and gains on 1p, 8q, 10q and 12q. Molecular alterations involving chromosome 9 were highly complex and copy number loss in the CDKN2A region was positively associated with deletions on 6p, 6q, 9p, 9q, 11p, 13q and 17p, and gains on 1q, 4q, 7p, 7q, 8p, 17q and 20q in MI tumors. Our findings add information to conventional histopathological classification and may lead to improved biological understanding and the development of robust prognostic biomarkers. Studies of larger numbers of tumors are underway to confirm the subgroups and relate findings to clinico-pathological data.

Citation Format: Carolyn D. Hurst, Fiona M. Platt, Claire F. Taylor, Margaret A. Knowles. Subgroups of urothelial carcinoma of the bladder defined by genomic analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 821. doi:10.1158/1538-7445.AM2013-821
Abstract 831: Elucidating the roles of differentially expressed microRNAs in glioblastoma multiform.
Authors: Liu Yi Shiuan
Published: August 2014

To understand the roles of miRNA in the pathogenesis of glioblastoma multiform (GBM), we used the quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay to profile 29 pairs of GBM core and peripheral tissue samples. We also employed deep sequencing technology to determine the expression levels of human miRNAs in one pair of GBM samples. These studies revealed that human miRNA miR-196b was over-expressed in GBM samples. Expression levels of miR-196b in cell lines derived from GBM tumors also exhibit a grade-dependent increase.

To explore the function of miR-196b in GBM, we constructed lentiviral vectors and overexpressed miR-196b in GBM cell lines. Over-expression of miR-196b enhanced cell growth and survival, promoted anchorage-independent growth and enhanced migration. To understand the targets and pathways underlying the tumor promoting activity of miR-196b, we compared gene expression patterns in control and miR-196b over-expressing cells. The cDNA microarray data were analyzed using the online GSEA Bioinformatics tools to indentify pathways altered by miR-196b. We also performed an in-house TargetScan analysis to predict and score putative miR-196b targets. Finally, results from GSEA analyses were combined with the target list which contained 700 high efficacy human miR-196b targets to identify targets of miR-196 with significant biological impact.

Through these studies, we will explore the potential roles of miR-196b in the regulation of tumor suppressor genes in brain cancer by elucidating the mechanisms of direct or indirect targets and downstream related genes.

Citation Format: Yi Shiuan Liu. Elucidating the roles of differentially expressed microRNAs in glioblastoma multiform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 831. doi:10.1158/1538-7445.AM2013-831

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 834: NRH:quinone oxidoreductase 2 (NQO2) stabilization of C/EBPα against 20S proteasomal degradation leads to protection against radiation-induced myeloproliferative diseases.
Authors: Xu Junkang , Jaiswal Anil
Published: August 2014

NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical induced oxidative stress. Disruption of NQO2 gene in mice leads to γ-radiation-induced myeloproliferative diseases. In this report, we found that NQO2 in presence of its cofactor NRH protected C/EBPα against 20S degradation. Coimmunoprecipitation studies showed that 20S proteasome and NQO2 both interact with myeloid differentiation factor C/EBPα. 20S proteasome interaction with C/EBPα led to the degradation of C/EBPα. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBPα. Exposure of mice and HL-60 cells to γ-radiation enhanced the levels of NQO2, which led to increased NQO2 interaction with C/EBPα and decreased 20S interaction with C/EBPα. This resulted in the stabilization of C/EBPα and protection against γ-radiation-induced myeloproliferative diseases. NQO2-/- mice deficient in NQO2 failed to stabilize C/EBPα. This contributed to the development of γ-radiation-induced myeoproliferative disease in NQO2-/- mice.

Citation Format: Junkang Xu, Anil Jaiswal. NRH:quinone oxidoreductase 2 (NQO2) stabilization of C/EBPα against 20S proteasomal degradation leads to protection against radiation-induced myeloproliferative diseases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 834. doi:10.1158/1538-7445.AM2013-834
Abstract 836: Decursin and doxorubicin are in synergy for the induction of apoptosis via STAT3 and/or mTOR pathways in human multiple myeloma cells.
Authors: Jung Duck-Beom , Jeong Soo-Jin , Kwon Hee-Young , Jung Ji Hoon , sohn Eunjung , Sin Eun Ah , Kim Ji-Hyun , Cho Sun-Mi , Yun Miyong , Lee Hyo-jung , Kim Sung Hoon
Published: August 2014

Drug resistance causes a serious limitation for the chemotherapy of multiple myeloma. Combination cancer therapy is one of attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin (a major compound in Angelica gigas) and doxorubicin on the induction of apoptosis in three human multiple myeloma cells. Concurrent treatment of decursin and doxorubicin significantly exerted significant cytotoxicity, while doxorubicin or decursin alone had no cytotoxic effect at physiologically relevant concentrations in signal transducer and activator of transcription 3 (STAT3) positive cell lines such as U266 and RPMI8226 cells and also STAT3 negative cell line MM.1S cells. We further demonstrated that the loss of the viability of the cells co-treated with decursin and doxorubicin was due to the induction of apoptosis. Combination treatment of doxorubicin and decursin enhanced the activation of caspapse-9 and 3, the cleavage of poly (ADP-ribose) polymerase (PARP) and sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment of doxorubicin and decursin downregulated the phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream S6K1 and activated the phosphorylation of extracellular signal regulated kinase (ERK) in three multiple myeloma cells. Furthermore, the combined treatment of doxorubicin and decursin reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3 and Src, activated SHP-2 and attenuated the expression of cyclind-D and survivin in STAT3 positive U266 cells. Conversely, the broadly-acting tyrosine phosphatases inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activatio induced by combine treatment of doxorubicin and decursin in U266 cells.

Citation Format: Duck-Beom Jung, Soo-Jin Jeong, Hee-Young Kwon, Ji Hoon Jung, Eunjung sohn, Eun Ah Sin, Ji-Hyun Kim, Sun-Mi Cho, Miyong Yun, Hyo-jung Lee, Sung Hoon Kim. Decursin and doxorubicin are in synergy for the induction of apoptosis via STAT3 and/or mTOR pathways in human multiple myeloma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 836. doi:10.1158/1538-7445.AM2013-836
Abstract 842: Role of Galectin-3 in invasive and apoptotic properties of neuroblastoma cells.
Authors: Wesley Umadevi V. , Resad Esat , Dempsey Robert J.
Published: August 2014

Background: Neuroblastoma (NB) is a childhood cancer derived from the neural crest stem cells. It generally progresses to an aggressive therapy resistant, fatal metastatic tumor. MYCN over-expression and amplification, and increased BDNF/TrkB signaling are used as signatures of high-risk NB. However, only about 25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. Up-regulation of Galectin 3 (Gal-3), a multifunctional, carbohydrate binding cytokine has been extensively correlated with highly malignant behavior and poor prognosis of many cancers. However, the role of Gal-3 in NB is largely unknown. In this study, we investigated the expression pattern of Gal-3 in a panel of NB cell lines and examined whether silencing of Gal-3 expression by means of a siRNA approach decreases their invasive properties, and increases their sensitivity to pro-apoptotic chemotherapeutic drug, fenretinide (4-HPR), a synthetic retinoid derivative.

Materials and Methods: We utilized RT-PCR gene expression analysis, RNA interference approaches, western blot and immunofluorescence analysis, scratch technique, transwell Boyden chamber migration assay, colony forming assay, and cytokine antibody array screening to address these studies.

Results: Gal-3 expression is differentially regulated in TrkB-positive, MYCN-amplified (SMS-KCNR and SK-N-BE) and TrkB-negative, non-MYCN-amplified (SK-N-AS, SK-N-SH, and SH-SY5Y) NB cell lines. Gal-3 expression was significantly higher in SK-N-AS cells that are of glial sub-lineage. Gal-3 was expressed both in cytoplasm and membrane. On the other hand, Gal-3 expression was relatively lower in less invasive, non-tumorigenic SK-N-SH cells. Silencing of Gal-3 by a small-interfering RNA against Gal-3 (Gal-3-SiRNA) decreased the colony forming potential, in vitro migratory and angiogenic abilities of SK-N-AS cells. Pre-treatment with Gal-3 SiRNA increased the sensitivity of SK-N-AS cells to 4-HPR induced apoptosis. Furthermore, exogenous Gal-3 treatment in neuroblast like Neuro-2a cells resulted in the up-regulation of IL-6 and phosphorylation of the pro-survival molecule AKT.

Discussion: Our data suggest that Gal-3 mediates migratory and invasive phenotype of non-MYCN amplified NB derived cells possibly via regulating the IL-6 signaling and Akt phosphorylation. Silencing of Gal-3 may be potentially useful to suppress the resistance of NB cells to apoptosis.

Citation Format: Umadevi V. Wesley, Esat Resad, Robert J. Dempsey. Role of Galectin-3 in invasive and apoptotic properties of neuroblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 842. doi:10.1158/1538-7445.AM2013-842
Abstract 843: Inhibition of AKT signaling promotes FOXO3a-dependent apoptosis in castration-resistant prostate cancer cells.
Authors: Das Trinath P. , Damodaran Chendil
Published: August 2014

Treatment for aggressive prostate cancers involves hormonal ablation therapy, radical prostatectomy, radiation therapy, chemotherapy or in combination. Despite their initial sensitivity to these treatments, prostate cancer eventually progresses to castration -resistant prostate cancer (CRPC), which is both aggressive and refractory to current therapeutic modalities. Hence, CRPC demands the identification of novel targeted therapies with minimal side effects. We identified Withaferin-A (WA) a major bioactive compound derived from the medicinal plant, Withania somnifera, is extensively used in Asian herbal medicines to treat a variety of ailments, including cancer. Earlier, we reported that WA induced the pro-apoptotic gene, prostate apoptosis response-4 (Par-4) and apoptosis in CRPC cells. These results encouraged us to determine the mechanism of action of WA on CRPC cells. It is known that activated AKT negatively regulates forkhead transcription factor class O family protein (FOXO3a) that regulates genes involved with tumorigenesis, cell cycle arrest or apoptosis.

In the present study, we found that FOXO3a is an upstream of Par-4 signaling which transactivates Par-4 function in CRPC cells. Inhibition of AKT activation by WA facilitates nuclear translocation of FOXO3a which in turn activates Par-4 mediated apoptosis in CRPC cells. Our confocal and immuno-precipitation studies confirmed that upon activation. FOXO3a and Par-4 were co-localized in the nucleus. Similar results were found with overexpression of FOXO3a showing transactivation of Par-4 and growth arrest in CRPC cells. Treatment of actinomycin-D or cyclohexmide along with WA, confirmed that FOXO3a regulation on Par-4 function in CRPC cells. On contrary, inhibition of FOXO3a by siRNA resulted in down regulation of Par-4 and apoptosis by WA in CRPC cells suggesting FOXO3a activation is necessary for the induction of Par-4 mediated apoptosis. Promoter bashing studies suggest a direct interaction of FOXO3a with Par-4 promoter; a sequential deletion of FOXO3a DNA binding sites in Par-4 promoter fails to induce Par-4 activation upon WA treatment in CRPC cells. Overall, our studies conclude that Par-4 is a target of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a is an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC.

Citation Format: Trinath P. Das, Chendil Damodaran. Inhibition of AKT signaling promotes FOXO3a-dependent apoptosis in castration-resistant prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 843. doi:10.1158/1538-7445.AM2013-843
Abstract 860: In vitro and in vivo studies of a novel, functionalized, completely human serine protease targeting Her2/neu. Activity against Herceptin- and multi-drug resistant tumor cells .
Authors: Cao Yu , Mohamedali Khalid A. , Marks John W. , Cheung Lawrence H. , Hittelman Walter N. , Rosenblum Michael G.
Published: August 2014

A number of recombinant fusion constructs and drug conjugates demonstrate excellent efficacy in preclinical studies but the long-term clinical applicability of these agents may be limited by tolerability issues and the emergence of multi-drug resistance. To address these issues, we employed the human serine protease granzyme B (GrB) as a cytotoxic effector and the humanized scFv designated 4D5 targeting the Her2/neu extracellular domain. A human fusion construct designated GrB/4D5/26 containing the fusogenic peptide 26 was constructed. The GrB/4D5/26 fusion construct showed specific and rapid cellular uptake into Her2/neu positive but not Her2/neu negative cells. We demonstrated efficient, specific delivery of GrB to the cytosol of Her2/neu positive cells. Against a panel of Her2/neu-positive tumor cells, IC50 values of the fusion construct ranged from 10∼100 nM, compared to that of GrB which was in the μM range. Treatment with GrB/4D5/26 resulted in specific cell killing accompanied by activation of caspases, PARP cleavage and cytochrome C release from mitochondria. In contrast to studies from other groups, the cytotoxic effects of the GrB/4D5/26 fusion construct did not require the assistance of the endosomolytic reagent chloroquine. Treatment with GrB/4D5/26 also caused efficient downregulation of the PI3K/Akt and Ras/ERK signaling pathways. Tumor cell lines highly resistant to Lapatinib, Herceptin or chemotherapeutic agents were not cross-resistant to the GrB-based fusion protein. Consecutive intravenous injections of GrB/4D5/26 into nude mice bearing BT474 M1 tumors resulted in significant, long-term tumor growth suppression. Twenty-four hours after i.v. administration, excised tumors displayed an increase in GrB/4D5/26 concentration and apoptotic nuclei compared to saline controls. These studies demonstrate the impressive in vitro and in vivo activity of the the completely human Her2/neu targeted construct and suggest its further evaluation for clinical use. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Yu Cao, Khalid A. Mohamedali, John W. Marks, Lawrence H. Cheung, Walter N. Hittelman, Michael G. Rosenblum. In vitro and in vivo studies of a novel, functionalized, completely human serine protease targeting Her2/neu. Activity against Herceptin- and multi-drug resistant tumor cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 860. doi:10.1158/1538-7445.AM2013-860
Abstract 862: Purified recombinant Maspin is processed through the endosomal pathway in breast cancer cells.
Authors: Bodenstine Thomas M. , Seftor Richard E. B. , Seftor Elisabeth A. , Khalkhali-Ellis Zhila , Samii Nicole A. , Monarrez J. Cesar , Pemberton Philip A. , Hendrix Mary J. C.
Published: August 2014

Maspin (Mammary Serine Protease Inhibitor), a gene involved in mammary gland development and morphogenesis, has been shown to act as a tumor suppressor in multiple cancers by inhibiting the aggressive cancer cell phenotype. Recombinant Maspin (rMas) added to cell culture media is internalized by cancer cells and capable of recapitulating many of the effects of endogenously re-expressed Maspin in Maspin-null cancer cells, including the inhibition of cell invasion, migration and induction of angiogenesis. Importantly, these effects are mediated by purified rMas without the need for post-translational modifications prior to application, increasing the future therapeutic potential for utilizing rMas as an adjuvant therapy. However, the therapeutic use of rMas is limited by its rapid clearance from cancer cells, and many of the molecular mechanisms regarding rMas internalization and processing remain to be determined. Building on preliminary data, we hypothesized that rMas is processed by the endosomal pathway. We demonstrate localization of rMas in Rab9 late-endosomes and Lamp-1 positive lysosomes following treatment of 10 μg/ml

rMas in BT549, Hs578T and MDA-MB-231 human breast cancer cell lines. rMas internalization was reduced when incubated at 4oC, and increased at temperatures of 20oC and 37oC, with the highest rates of internalization at 37oC, indicative of endocytosis. Furthermore, depletion of K+ decreased rMas internalization in Hs578T and

MDA-MB-231, suggestive of clarthrin-mediated endocytosis. Our data show that

rMas is capable of acting at the membrane to mediate effects on invasion and migration, but may not be functional once internalized due to rapid degradation by the lysosome. Interestingly, this may provide a plausible explanation for previously reported data in which rMas was not able to reproduce the effects of endogenous Maspin re-expression on cancer cell apoptosis sensitivity, which require cytosolic and mitochondrial mechanisms. Our data provide evidence that strategies which induce endosomal escape of rMas will intensify its anti-tumor effects on cancer cells, and further enhance its potential therapeutic applications.

Citation Format: Thomas M. Bodenstine, Richard E. B. Seftor, Elisabeth A. Seftor, Zhila Khalkhali-Ellis, Nicole A. Samii, J. Cesar Monarrez, Philip A. Pemberton, Mary J. C. Hendrix. Purified recombinant Maspin is processed through the endosomal pathway in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 862. doi:10.1158/1538-7445.AM2013-862
Abstract 864: ANG4043: A new brain-penetrant peptide-antibody conjugate that reduces tumor growth in a HER2-positive orthotopic tumor model.
Authors: Lachowicz Jean E. , Demeule Michel , Che Christian , Tripathy Sasmita , Currie Jean-Christophe , Regina Anthony , Nguyen Tran , Lord-Dufour Simon , Castaigne Jean-Paul
Published: August 2014

While monoclonal antibodies to receptor tyrosine kinases such as HER2 have been demonstrated to reduce tumor size and increase survival, these agents achieve little to no brain penetration, making them ineffective against metastatic brain tumors. The blood-brain barrier (BBB), which restricts entry of proteins such as mAbs into the brain, is comprised of capillary endothelial cells with tight junctions and efflux pumps. Brain entry of nutrients, hormones, and other required molecules is accomplished by processes such as receptor-mediated transcytosis. As low-density lipoprotein receptor-related protein 1 (LRP1) is known to perform this function in BBB endothelial cells, we have created a family of peptides (Angiopeps) designed for LRP1 recognition. These proprietary Angiopeps can be used to create novel Peptide-Drug Conjugates that successfully cross the BBB by LRP1-mediated transcytosis. Here we describe chemical conjugation of Angiopep-2 (An2) to a mAb against HER2. This Peptide-Antibody Conjugate, ANG 4043, displays HER2 binding affinity and in vitro cytotoxic potency similar to that of native anti-HER2. ANG4043 demonstrates a high rate of entry into the brain, consistent with achieving therapeutic concentrations. The plasma half-life of ANG4043 is similar to that of Anti-HER2 in mouse. Mice with intracranially implanted BT-474 cells showed reduced brain tumor size when dosed with ANG4043 compared to controls. Overall, these data demonstrate that a brain-penetrant Peptide-Antibody Conjugate is efficacious in a mouse HER2-positive tumor model. These results extend the validation of An2 conjugation beyond small molecules and peptides to include larger molecules such as therapeutic mAbs for development of new brain-penetrant antitumor therapeutics.

Citation Format: Jean E. Lachowicz, Michel Demeule, Christian Che, Sasmita Tripathy, Jean-Christophe Currie, Anthony Regina, Tran Nguyen, Simon Lord-Dufour, Jean-Paul Castaigne. ANG4043: A new brain-penetrant peptide-antibody conjugate that reduces tumor growth in a HER2-positive orthotopic tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 864. doi:10.1158/1538-7445.AM2013-864
Abstract 867: IGF-I receptor targeting therapy for esophageal carcinomas.
Authors: Adachi Yasushi , Yamamoto Hiroyuki , Ohashi Hirokazu , Matsunaga Yasutaka , Nosho Katsuhiko , Suzuki Hiromu , Taniguchi Hiroaki , Arimura Yoshiaki , Endo Takao , Kato Yasuo , Imai Kohzoh , Carbone David P. , Shinomura Yasushisa
Published: August 2014

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for tumorigenicity and cancer development of many malignancies, however this pathway has not been well studied in human esophageal carcinomas. Esophageal cancer is one of worst prognostic disease, main types of which are squamous cell carcinomas (ESCC) and adenocarcinoma (EAC). Previously we have reported that the IGF axis may play a key role in tumor progression of ESCC and its detection may be useful for the prediction of recurrence and poor prognosis and possibly for selecting patients for targeting therapy for IGF-IR. We have previously shown successful therapy for several gastrointestinal cancers using recombinant adenoviruses expressing dominant negative forms of IGF-IR (Ad-IGF-IR/dn). In this study, we wanted to develop potential targeted therapeutics to IGF-1R; therefore, we assessed the effect of IGF-IR blockade in both types of esophageal cancer. We analyzed immunohistochemical expression of IGF-IR in a tissue array. A tyrosine kinase inhibitor, BMS-536924, and Ad-IGF-IR/dn were used to treat several ESCC lines, such as TE1, TE8, TT, TTn, and EAC lines, including OE19 and OE33. We assessed the effect of both IGF-IR blockade on signal transduction, growth, and apoptosis. IGF-IR is expressed frequently in esophageal tumors, but not in normal mucosa. IGF-IR was produced in ESCC more than EAC, and is detected in metastasized similar to the primary site. IGF-IR inhibition suppressed proliferation and colony formation in both types of cancer. Blockade of IGF-IR up-regulated both stress- and chemotherapy-induced apoptosis in both esophageal tumor type. Although IGF-IR/dn blocked ligand-induced activation of Akt-1 mainly, BMS-536924 effectively blocked both activation of Akt and MAPK. Thus, the IGF axis may play a key role in tumor progression of human esophageal carcinomas. The IGF-IR targeting strategies might be useful anticancer therapeutics which can be used widely for human esophageal malignancies.

Citation Format: Yasushi Adachi, Hiroyuki Yamamoto, Hirokazu Ohashi, Yasutaka Matsunaga, Katsuhiko Nosho, Hiromu Suzuki, Hiroaki Taniguchi, Yoshiaki Arimura, Takao Endo, Yasuo Kato, Kohzoh Imai, David P. Carbone, Yasushisa Shinomura. IGF-I receptor targeting therapy for esophageal carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 867. doi:10.1158/1538-7445.AM2013-867
Abstract 874: Mutations of aromatic amino residue to polar residue in NBDs alter the transport properties of ABCC10.
Authors: Malofeeva Ekaterina V. , Hopper-Borge Elizabeth
Published: August 2014

ABCC10 is an efflux pump that confers multidrug resistance to cells by extruding a variety of natural and nucleosides analogues using energy from ATP hydrolysis. The objective of this project is to understand the detailed relationship between ATP hydrolysis and drug transport for ABCC10 and how ABCC10’s ATPase activity is regulated. For this study, we mutated aromatic residues to polar residues in the Nucleotide Binding Domains (NBDs) of ABCC10 to determine how these residues are involved in the transport of ABCC10 substrates. Prior, structural analyses of several bacterial ABC-transporters indicated that aromatic amino acid residues in NBDs are needed for ATP binding and ATP hydrolysis. In these studies, substitution of these aromatic residues completely abolished ATP-dependent transport. Prior work on ABCC1 has shown that substitutions such as W (tryptophan) to C (cytidine) or Y (tyrosine) to C (cytidine) decreased ABCC1’s affinity for ATP, and ATP-dependent LTC4 transport activities. Analogous substitutions of W (tryptophan) residues to polar residues in the NBD2 of ABCC2 eliminated estradiol glucuronide and leukotriene C4 transport. Based on these data, we hypothesize that mutating aromatic to polar residues in the NBDs of ABCC10 will modulate the biochemical and cellular transport activities of this efflux pump.

At present we have generated mutant-ABCC10 constructs by site-directed mutagenesis. Specifically, we mutated the aromatic Tyr747 (NBD1), Trp607 (NBD1) and Tyr1255 (NBD2) residues to polar cysteine residues. We then examined all ABCC10-mutant, wild-type ABCC10 and empty-vector constructs using immunofluorescence, ATPase assays, sensitivity and transport assays. We observed that the mutant ABCC10 proteins localized predominantly to the membranes. Further, ABCC10-mutant cells, except for the W607C mutant clone, showed less sensitivity than empty vector-transfected cells. Using accumulation assays we also demonstrated that substitutions of aromatic to polar residues, such as W607C and Y1255C, decreased the ability of ABCC10 to transport, docetaxel and Ara-C, however transport was not completely abolished. Using our ABCC10 mutants we found that aromatic to polar residue substitutions eliminated the stimulatory effects of docetaxel and Ara-C on ATPase activity of ABCC10.

Taken together, these results demonstrate that these aromatic residues are involved in the ATP binding process and mutations of these residues cause of reduction in ABCC10 transport of docetaxel and Ara-C from cells.

Citation Format: Ekaterina V. Malofeeva, Elizabeth Hopper-Borge. Mutations of aromatic amino residue to polar residue in NBDs alter the transport properties of ABCC10. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 874. doi:10.1158/1538-7445.AM2013-874
Abstract 876: Molecular mechanisms of paclitaxel-resistance and resveratrol sensitivity in MDA-MB-231 breast cancer cells.
Authors: Sprouse Alyssa A. , Herbert Brittney-Shea
Published: August 2014

Treatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. To study cancers that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the breast cancer cell line MDA-MB-231. A “spiking” method of paclitaxel treatment was used to select for a population of cells that are resistant to the drug. This method mimics the development of resistance in recurrent tumors in patients. The IC50 of paclitaxel in these cells was 75 μM compared to the 0.037 μM IC50 of the parent cell line, a 2000-fold increase in resistance. In this very heterogeneous population, the mechanism of resistance was not due to increased protein levels of the efflux protein, p-glycoprotein, as quantitated by western blot analysis. To better study these cells, the paclitaxel-resistant cell line was cloned using a limiting dilution method to provide more homogeneous populations of resistant cells. The 29 clones obtained exhibited a paclitaxel IC50 range of 8 μM to 78 μM which was equivalent to a 200- to 2000-fold increase in resistance compared to the parent line. It has been suggested that the polyphenol natural compound, resveratrol, which has been shown to inhibit cell growth of multiple cancer types, may be useful as a combination anti-cancer treatment or novel therapeutic for drug-resistant cancer cells. There was no significant difference among the 72 hour IC50 of resveratrol in the parent line, the heterogeneous resistant line, the least paclitaxel-resistant clone or the most paclitaxel-resistant clone. We observed that treatment with 10-100 μM concentrations of resveratrol in all cell lines showed a reduction in cell proliferation and increased apoptosis within 72 hours (p<0.05), with the paclitaxel-resistant cells to a greater extent. In addition, resveratrol decreased the ability of the parent, heterogeneous resistant and the highest resistant clone cells to form colonies (an indication of reduced cell survival capacity). To better characterize the molecular mechanisms of the paclitaxel-resistant cell line and the effects of resveratrol treatment, RNA sequencing will reveal the unique transcriptome and pre-miRNA changes in the resistant cells compared to the parent as well as changes in both cell lines due to resveratrol treatment. This resistant cell line and its clones provide a powerful tool to study paclitaxel-resistance in and therapeutics for breast cancer.

Citation Format: Alyssa A. Sprouse, Brittney-Shea Herbert. Molecular mechanisms of paclitaxel-resistance and resveratrol sensitivity in MDA-MB-231 breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2013-876
Abstract 877: Efficacy of Salmonella typhimurium A1-R on chemoresistant stem-like human pancreatic cancer cells.
Authors: Hiroshima Yukihiko , Zhao Ming , Zhang Yong , Maawy Ali , Hassanein Mohamed , Suetsugu Atsushi , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M.
Published: August 2014

We have previously developed the genetically-modified Salmonella typhimurium A1-R (A1-R) strain that selectively targets tumors. We have also previously reported that XPA1 human pancreatic cancer cells are dimorphic (spindle and round shape), with the spindle-shaped cancer cells having the greater capability for distant metastasis, suggesting they are stem-like cells. The aim of this study is to examine whether spindle-shaped XPA1 cells are chemo-resistant and if they are sensitive to A1-R. Serial dilution was performed to obtain clones of both cellular morphologies. To determine chemosensitivity, in vitro IC50 values were obtained for both types of XPA1 cells for (1) Cisplatin (CDDP); (2) 5-fluorouracil (5FU); (3) Gemcitabine (GEM); and (4) A1-R. The efficacy of these drugs was also determined for both types of XPA1 cells in vivo. IC50 values of spindle-shaped and round XPA1 cells, respectively, were 2.65 ± 0.22 μg/ml and 1.43 ± 0.36 μg/ml for CDDP (p = 0.012); 2.44 ± 0.25 μg/ml and 1.48 ± 0.19 μg/ml for 5FU (p = 0.007); 3.17 ± 0.15 ng/ml and 2.70 ± 0.29 ng/ml for GEM (p = 0.133); and (19.7 ± 1.46) x 108 cfu/ml and (17.8 ± 9.78) × 108 cfu /ml for A1-R (p = 0.771). In vivo, 5FU and A1-R significantly reduced the tumor weight of round cells (5FU; p = 0.028; A1-R; p = 0.011). However, only A1-R significantly reduced the tumor weight of spindle-shaped cells (p = 0.012). The spindle-shaped XPA1 cells were thus chemo-resistant and sensitive to A1-R, suggesting A1-R could be a promising therapy for chemo-resistant human pancreatic cancer stem-like cells.

Citation Format: Yukihiko Hiroshima, Ming Zhao, Yong Zhang, Ali Maawy, Mohamed Hassanein, Atsushi Suetsugu, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Efficacy of Salmonella typhimurium A1-R on chemoresistant stem-like human pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 877. doi:10.1158/1538-7445.AM2013-877
Abstract 881: Hyperthermic isolated intraperitoneal chemoperfusion (HIPEC) for treatment of patients with peritoneal carcinosis - Analysing negative influencing cellular mechanisms in the tumor cells.
Authors: Gasser Martin , Lazariotou Maria , Vetterlein Malte , Grimmig Tanja , Germer Christoph T. , Pelz Joerg , Waaga-Gasser Ana Maria
Published: August 2014

Background: In patients with isolated peritoneal carcinosis (PC) of gastric, colorectal and ovarian cancer hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment option integrated into multimodal concepts. We analyzed relevant heat shock proteins (HSPs) that confer resistance to physical stress like hyperthermia as well as specific multidrug resistence (MDR) genes in patients with PC.

Materials/Methods: Patients with different adenocarcinomas and additional PC that underwent HIPEC therapy between 09/09 and 03/11 in our department (gastric, colorectal and ovarian cancer) were included in the study. HIPEC therapy was performed under specific conditions (1hr permanent chemotherapeutical flux via external pump into the abdominal cavity after resection of relevant tumor masses with elevated temperature up to 43°C). Tumors before and after HIPEC therapy were analyzed for HSPs, ABC transporter and CD133 expression by immunohistochemistry, Western Blot, and RT-qPCR. Additionally, HT29 tumor cells were exposed in vitro to different conditions of hyperthermia up to 43°C for 60 minutes and comparably analyzed. Tumor cells were counted and studied for apoptosis.

Results: HSP70/72, HSP90 and CD133 expression was upregulated in the investigated tumors, in particular post HIPEC therapy. Upregulated protein and gene expression was also shown for MDR genes ABCB5, ABCG2 and ABCC5. Hyperthermia induced upregulated protein and gene expression in HT29 tumor cells dependent on incubation temperature, mainly for HSPs observed in western blot, immunohistochemistry and RT-qPCR. Furthermore, cell viability decreased with increasing incubation temperature.

Conclusion: Therapeutic approaches like HIPEC to achieve antiproliferative and apoptosis inducing cellular effects in patients with PC seem to be negatively influenced by highly conserved HSP mechanisms as well as multidrug resistance genes. Studying HSP and MDR expression profiles both in patient tumors and in vitro are valuable tools to further clarify the effects of hyperthermia and chemotherapeutical agents in context with the treatment of HIPEC therapy in the affected patients.

Citation Format: Martin Gasser, Maria Lazariotou, Malte Vetterlein, Tanja Grimmig, Christoph T. Germer, Joerg Pelz, Ana Maria Waaga-Gasser. Hyperthermic isolated intraperitoneal chemoperfusion (HIPEC) for treatment of patients with peritoneal carcinosis - Analysing negative influencing cellular mechanisms in the tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 881. doi:10.1158/1538-7445.AM2013-881
Abstract 882: Functional investigation of a novel cancer specific organic anion transporting polypeptide 1B3 variant 1 (OATP1B3 V1) in colon and pancreatic cancer.
Authors: Thakkar Nilay , Kim Kyungbo , Jang Eun Ryoung , Han Songhee , Kim Kyunghwa , Kim Donghern , Merchant Nipun , Lockhart A. Craig , Lee Wooin
Published: August 2014

OATP1B3 is a member of the Organic Anion Transporting Polypeptide (OATP) superfamily that mediates the uptake of numerous endogenous and xenobiotic compounds. Initially, OATP1B3 was reported to be expressed exclusively in the normal hepatocytes. Subsequent studies have reported that OATP1B3 is frequently expressed in multiple types of cancer tissues derived from gastrointestinal tract, pancreas, prostate, lung and breast. Some reports have also suggested that OATP1B3 may be associated with differing clinical outcomes. However, so far the biological functions of OATP1B3 in cancer have not been elucidated. In this study, we confirmed that colon and pancreatic cancer cells express variant forms of OATP1B3, different from OATP1B3 wildtype (WT) expressed in normal liver. OATP1B3 variant 1 (V1) is the most prevalent form among the variants containing the alternate exon 2a, instead of exons 1 and 2 as in OATP1B3 WT. The translated protein sequence of OATP1B3 V1 is similar to OATP1B3 WT except lacking the first 28 amino acids at the N-terminus. OATP1B3 V1 has a limited transport activity toward cholecystokin-8 (CCK-8, a prototype OATP1B3 substrate) in contrast to OATP1B3 WT which showed a markedly efficient uptake of CCK-8. Consistent with these results, OATP1B3 V1 was localized mainly in the cytoplasm with a much lower extent of trafficking to the surface membrane compared to OATP1B3 WT. In summary, our results demonstrate that colon and pancreatic cancer cells express variant forms of OATP1B3 with a limited transport activity and different subcellular localization compared to OATP1B3 WT. These observed differences at molecular and functional levels will be important considerations for further investigations of the biological and clinical significance of OATP1B3 expression in cancer.

Citation Format: Nilay Thakkar, Kyungbo Kim, Eun Ryoung Jang, Songhee Han, Kyunghwa Kim, Donghern Kim, Nipun Merchant, A. Craig Lockhart, Wooin Lee. Functional investigation of a novel cancer specific organic anion transporting polypeptide 1B3 variant 1 (OATP1B3 V1) in colon and pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 882. doi:10.1158/1538-7445.AM2013-882