bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2475: Effect of mTOR inhibition by rapamycin on promotion of skin tumors in BK5. Aktwt transgenic mice
Authors: Rho Okkyung , Jiang Guiyu , Sandifer Allyson , DiGiovanni John
Published: February 2011

Akt is a serine/threonine kinase involved in a variety of cellular responses including cell survival and proliferation. It has been shown that PI3K/Akt signaling is frequently altered (upregulated) in human cancers. mTOR is one of the Akt downstream signaling pathways that regulates protein synthesis, cell growth and proliferation. mTOR exists in two complexes, mTORC1 and mTORC2. Our laboratory previously reported that tumor promoters including TPA, okadaic acid (OA), and chrysarobin (CHRY) activate epidermal Akt after topical treatment of mouse epidermis and mouse primary cultured keratinocytes. Activation of Akt during tumor promoter treatment leads to enhanced downstream signaling including mTORC1 pathways. BK5. Aktwt transgenic mice where Akt is overexpressed in basal epidermis were significantly more sensitive to two-stage carcinogenesis, showing tumors with a much shorter latency, considerably larger in size, and enhanced progression of papillomas to SCCs. Importantly, Akt transgenic mice showed significant elevations in several downstream signaling pathways both in the absence and presence of TPA, including mTORC1 activity. Collectively, the studies with transgenic mice overexpressing Akt in epidermis (BK5.Aktwt) have suggested that activation of mTORC1 signaling may contribute to the process of skin tumor promotion. In this study, we have examined the ability of rapamycin, a known inhibitor of mTOR, on mTORC1 signaling in relation to its effect on TPA-induced epidermal cell proliferation and skin tumor promotion. Rapamycin given at doses of 1 μmol, 100 nmol, 20 nmol, and 5 nmol 30 min prior to 6.8 nmol TPA treatment inhibited mTORC1 activity in a dose-dependent manner. Notably, in a multiple treatment regimen (4 applications of rapamycin and TPA over a two week period) high doses of rapamycin significantly inhibited TPA-induced Akt phosphorylation at serine 473 as well as mTORC1 signaling in a dose dependent way, suggesting mTORC2 inhibition induced by prolonged high dose rapamycin treatment. The ability of rapamycin (at doses of 100, 20, and 5 nmol per mouse) to inhibit skin tumor promotion by TPA in BK5. Aktwt transgenic mice was compared with wild-type mice. Rapamycin significantly reduced TPA promotion in BK5.Aktwt mice, however, it was less effective compared to similar doses used in wild-type mice. The data demonstrate that rapamycin, although less effective, still retained significant inhibitory activity toward TPA promotion even when Akt and mTOR activities were elevated. These data demonstrate an important role for mTOR signaling in skin tumor promotion of TPA.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2475.
Abstract 2480: Role of Stat1 in epithelial carcinogenesis
Authors: Bozeman Ronald , Abel Erika , DiGiovanni John
Published: February 2011

Aberrant expression and/ or activity of STATs 1, 3, and 5 have been reported in multiple human cancers as well as in murine models of tumorigenesis. Previous work performed in our lab demonstrated a critical role for Stat3 in mediating the initiation and promotion stages of epithelial multi-stage carcinogenesis in mouse skin. In light of these findings, we have begun investigating the role of other STATs, particularly Stat1, in multistage skin carcinogenesis using both wild-type and Stat1 knockout (KO) mice. Western blot analyses revealed that topical application of the skin tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and chrysarobin induced phosphorylation of Stat1 in mouse epidermis. In addition, following treatment with chrysarobin, the level of total Stat1 protein was also dramatically elevated. To further investigate the role of Stat1 during epithelial carcinogenesis, Stat1 KO mice and wild-type littermates were initiated with 25 nmol DMBA and promoted with either 6.8 nmol TPA or 220 nmol chrysarobin. No significant differences were observed in either tumor incidence or multiplicity when TPA was used as the promoting agent, suggesting that Stat1 does not play a significant role in TPA-mediated skin tumor promotion. In contrast, Stat1 KO mice exhibited a dramatic reduction in both tumor multiplicity and incidence as compared to wild-type littermates when chrysarobin was used as the promoting agent. Additionally, following topical treatment with chrysarobin, Stat1 KO mice had a reduced epidermal proliferative response. To further investigate the impact of Stat1 deletion on chrysarobin-induced tumor promotion, Stat1 KO mice and wild-type controls were treated topically with chrysarobin and the inflammatory response was assessed. Stat1 KO mice displayed a significant reduction in Cox-2 induction compared to wild-type controls. Preliminary findings also showed that Stat1 KO mice have reduced immune cell influx following chrysarobin treatment. To investigate the potential role of reactive oxygen species (ROS) in the effects of chrysarobin on Stat1 activation, the dorsal skin of FVB mice were pre-treated topically with the anti-oxidants ascorbyl palmitate and α-tocopherol acetate. Topical application of α-tocopherol acetate significantly attenuated the epidermal proliferative response and Stat1 activation suggesting that chrysarobin-mediated generation of ROS may play a role in its effects on Stat1 during tumor promotion. Additional studies are underway to further determine the mechanistic basis for the role of Stat1 in chrysarobin-mediated tumor promotion. Supported by NIH grant CA 76520.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2480.
Abstract 2483: Transgenic mice to evaluate the role of S-adenosylmethionine decarboxylase in skin carcinogenesis
Authors: Shi Chenxu , McCloskey Diane E. , Pegg Anthony E. , Feith David J.
Published: February 2011

To evaluate the role of S-adenosylmethionine decarboxylase (AdoMetDC) and altered polyamine levels in mouse skin carcinogenesis, we utilized the tetracycline-regulated system to produce conditional expression of AdoMetDC in basal keratinocytes. AdoMetDC is a key enzyme in polyamine biosynthesis and it catalyzes the decarboxylation reaction converting AdoMet to dcAdoMet. dcAdoMet provides the aminopropyl groups for the synthesis of the higher polyamines spermidine and spermine from the precursor putrescine. Therefore, elevated AdoMetDC activity may promote the synthesis of higher polyamines at the expense of putrescine, and increased putrescine levels are tightly linked to tumor promotion in mouse skin. AdoMetDC activity is also known to be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). To make the DNA construct for microinjection, human AdoMetDC cDNA was inserted into the TetO vector followed by an internal ribosome entry site and luciferase coding sequence. Potential founder mice were crossed to mice with skin-specific expression of tetracycline-regulated transcriptional activator (tTA) directed by bovine keratin 5 (K5) promoter elements to generate K5-tTA/TetO-AdoMetDC bitransgenic mice. Transgene expression has been characterized by in vivo bioluminescent imaging of luciferase activity, in vitro luciferase activity as well as AdoMetDC activity assay and Western blotting in 7 week old mice. In bitransgenic mice, AdoMetDC is constitutively expressed in the skin and expression can be repressed by exposure to the tetracycline analogue doxycycline (Dox). In untreated bitransgenic mice generated from two founder lines, we saw a massive increase in luciferase activity and these mice have a thin fur phenotype. Minimal transgene expression is detected in either single transgenic TetO-AdoMetDC or Dox-treated bitransgenic mice. Untreated bitransgenic mice from one founder line exhibit a 7.2- fold increase in AdoMetDC activity in epidermis, a 7.8-fold increase in dermis, and dcAdoMet levels are also elevated by 8.0-fold in dermis. Effects of AdoMetDC overexpression on TPA induced polyamine biosynthesis and susceptibility to 7,12-dimethylbenz(a)anthracene/TPA carcinogenesis are now being investigated. These mice with regulated AdoMetDC expression in the skin provide a valuable model system to evaluate the role of AdoMetDC and polyamines in skin carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2483.
Abstract 2488: γ-Secretase inhibitor blocks cell proliferation in gastric cancers through mitotic arrest and apoptotic cell death
Authors: Shin Ji Young , Yi HwaJung , Chun Kyung-Hee
Published: February 2011

Gastric cancer is one of the most common cancers with mortal malignancy. Most patients with advanced gastric cancer have been treated with anti-cancer chemotherapies such as fluorouracil (5-FU), cisplatin and anthracycline which block mitotic cell division and induce consequent apoptotic cell death. However, there are needed novel gastric therapies and/or develop the new agents to overwhelm the resistance for traditional chemotherapeutic agents. Recent studies have reported that the Notch signaling pathway is associated with human gastric cancer and other cancers. Notch receptors are transmembrane proteins which are cleaved by protelolytic emzymes (gamma-secretase) and release their intracellular domain (ICD) upon activation status by ligand binding. In previous reports, notch intracellular domain (NICD) turns on target genes which are related with tumorigenesis such as COX-2, c-Myc and Snail in cancers, including gastric cancer cells. Thus we speculated that the blockage of notch signal would be useful strategy for chemotherapy of gastric cancers. We initially showed that Notch receptors and ligands are expressed in various gastric cancer cells through RT-PCR and western blotting. We also confirmed that Notch signals are activated in gastric cancer cells by Notch reporter gene analysis. Furthermore, GSI treatment reduced cell survival rate in gastric cancer cells, including AGS, SNU1, SNU601, and SNU638 cells. We observed G2/M mitotic arrest and subG1 arrest by GSI treatment in gastric cancer cells, accompanying with the increase of mitotic gene cyclin B1, cdc2, p21, and apototic caspase-3 activation. To demonstrate the effect of p21 increasing by GSI treatment, cell proliferation and G2/M cell cycle arrest was inhibited by GSI treatment in Myr-AKT lentivirus infection cell. Also, p21 and cyclinB1 protein levels were not increased after rapamycin treatment in Myr-AKT lentivirus infection cell. Collectively, these results suggest that GSI, which block Notch signal, would be a potential therapeutic drug in gastric cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2488.
Abstract 249: A Sleeping Beauty screen in mice identifies Zmiz1 as a candidate cancer gene in human squamous cell carcinomas
Authors: Rogers Laura M. , Swick Brian L. , Meyerholz David K. , Dupuy Adam J.
Published: February 2011

Cutaneous squamous cell carcinoma (SCC) is the second most frequent cancer in the United States, with over 250,000 new cases diagnosed annually. Current treatments rely upon surgical excision of the cancerous tissue. Often, however, surgical removal can be difficult for patients because tumors commonly develop on sun-exposed surfaces such as the face, ears, and hands. In addition, a low percentage of patients exhibit aggressive metastases that are not easily removed and often result in death. A deeper understanding of the underlying genes involved in disease initiation and progression will be essential for the development of nonsurgical treatments. A recent Sleeping Beauty (SB) transposon insertional mutagenesis screen identified Zmiz1 as a candidate cancer gene in cutaneous SCCs. Intriguingly, over 90% of SB-induced skin SCCs harbored mutations within the Zmiz1 gene, predicted to cause overexpression of an N-terminally truncated protein, which we named Zmiz1mut. Immunohistochemistry (IHC) analysis of SB-induced SCCs revealed aberrant nuclear Zmiz1 localization in tumor samples, while nuclear staining was absent in normal mouse skin. Importantly, nuclear Zmiz1 was also detected in ∼70% of human SCC tumors analyzed by IHC, indicating that aberrant Zmiz1 expression may also play a role in human disease. Furthermore, we have discovered that Zmiz1 nuclear staining is present in a significant percentage of breast, colon, and ovarian cancers.

The purpose of this study is to determine the functional significance of Zmiz1 mutation in cutaneous SCC. To this end, we engineered transgenic mice that express Zmiz1mut in a Cre-dependent manner. We crossed these mice to a K14-Cre strain to induce Zmiz1mut expression in the skin. Offspring inheriting both alleles proved to be tumor-prone, with over 80% of these animals developing SCC. These data confirm that Zmiz1 has oncogenic potential in the skin. Sequencing analysis of cDNA from tumors produced by our Zmiz1mut transgenic model uncovered no mutations in either p53 or H-Ras_mutations that are observed in other mouse models of skin cancer. IHC was also performed on tumor sections. As before, we observed a strong nuclear staining pattern in the tumors induced by Zmiz1mut expression, leading us to hypothesize that nuclear accumulation of Zmiz1 plays an active role in tumorigenesis. Current efforts are focused on further characterization of our unique skin cancer model and discovering the driving force behind nuclear accumulation of Zmiz1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 249.
Abstract 250: Vinculin is a putative amplification target gene at 10q22 in advanced prostate cancer
Authors: Ruiz Christian , Gonzales Irma M. , Oeggerli Martin , Schneider Sandra , Kiefer Jeffrey A. , Mousses Spyro , Barrett Michael T. , Azorsa David O. , Bubendorf Lukas
Published: February 2011

Patients with advanced prostate cancer are usually treated with androgen withdrawal. Although this therapy is effective at the beginning, nearly all prostate cancers become refractory to it. Approximately 15% of these hormone-refractory prostate cancers contain a genomic amplification at 10q22. Aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving gene.

We applied high-resolution array-CGH using the 244k Agilent microarrays to cell lines harboring 10q22 amplification. We identified a common amplified region (CAR) and silenced each of the 26 genes in this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22rv1. Genes with a significant growth reduction in the 10q22 amplified cell line PC-3 but not in the non-amplified 22rv1 cells were selected as putative candidate genes of this amplicon. They were further investigated in vivo by functional assays and in vitro by immunohistochemical analysis of the protein expression in more than 500 human prostate cancers on a tissue microarray (TMA).

We could narrow down the CAR to a region of 5.8 Mb. The siRNA screening experiments revealed Vinculin (VCL) as the most promising candidate gene of this amplicon. Immunohistochemical analysis of the Vinculin protein expression on a TMA enriched for 10q22 amplified prostate cancers showed a strong association between VCL gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that Vinculin expression was highest in hormone-refractory prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Notably, high tumor cell proliferation measured by Ki67 expression was significantly associated with high Vinculin expression in prostate cancer (p < 0.0001). Although there are countless reports on Vinculin as a cytoskeletal protein, its role in prostate cancer has previously not been investigated. Our data strongly suggest Vinculin as a major driving gene of the 10q22 amplification in prostate cancer and that Vinculin overexpression might contribute to prostate cancer progression by enhancing tumor cell proliferation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 250.
Abstract 2513: Arsenic trioxide induces depolymerization of microtubules in an acute promyelocytic leukemia cell line
Authors: Min Young Joo , Cha Seung Joo , Lee Hee-Soon , Baek Jin Ho , Park Min Jae , Kim Hawk , Park Jae-Hoo , Park Keon Uk , Won Jong-Ho
Published: February 2011

Arsenic trioxide (ATO) treatment has been used in recent decades to achieve clinical remission in patients with acute promyelocytic leukemia (APL). The biological basis for ATO treatment has been explained as being mediated through anti-proliferation, anti-angiogenesis, and apoptosis in both APL and solid tumor cells. Arsenic trioxide has been shown to affect microtubules such that dysfunction of the cellular cytoskeleton is induced, which ultimately leads to cell death. In this study, we reported that ATO disrupts tubulin assembly and results in microtubule dysfunction, inducing cell death in an APL cell line. ATO markedly enhanced the depolymerization of microtubules. Analysis of posttranslational modifications of microtubules also revealed that ATO decreased modified forms of the polymerized tubulin portion in adose-dependent manner. Immunocytochemical investigation showed that ATO changed the cellular microtubule network and inhibited the formation of polymerized microtubules. These alterations in microtubules suggest that ATO increases the depolymerized form of tubulin in the cells, potentially through its effect on the attenuation of spindle dynamics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2513.
Abstract 2517: Pharmacodynamic biomarkers for ARQ 736, a small molecule BRAF inhibitor
Authors: Yu Yi , Zhao Xiaolan , Gu Xiubin , Chang Edward , Cousens Laura , Chiesa Erin , Lowe Deirdre , Liu Yanbin , Bull Cathy O. , Waghorne Carol G. , Chan Thomas C.K.
Published: February 2011

The MAPK/ERK pathway is a cascade of serine/threonine protein kinases activated by various extracellular factors to control cell growth, differentiation, migration and death. A number of somatic mutations occur that lead to constitutive activation of this pathway in multiple human cancers. BRAF mutations were detected in over 60% of melanomas and may account for a significant proportion of colon cancer patients whose tumors are resistant to anti-EGFR therapy. ARQ 736 is a novel inhibitor of BRAF and is selectively potent in killing cancer cell lines harboring at least one mutated BRAF allele. The objective of this study is to identify potential protein biomarkers for use in connection with ARQ 736 treatment. A number of proteins released by BRAF (V600E) mutant A375 human melanoma cells into conditioned media detected by antibody arrays (R&D human angiogenesis array ARY007 and human cytokine array ARY005) are reduced following exposure to ARQ 736. These effects were not seen in the ARQ 736-resistant cell line DLD1, a k-ras mutant colon carcinoma line. Potential protein biomarkers identified include GM-CSF, Serpin E1, VEGF, amphiregulin, IGFBP-2, IGFBP-3, pentraxin-3 and IL-8. To investigate whether changes in secreted proteins observed in vitro in response to ARQ 736 treatment could be recapitulated in vivo, a similar analysis of plasma was performed at the termination of an A375 human tumor xenograft study. In this study, ARQ 736 administered intraperitoneally at 300 mg/kg on a Q1Dx13 schedule resulted in a 54% inhibition of growth of A375 human melanoma tumor xenografts. Constant administration of ARQ 736 by Alzet osmotic mini-pumps resulted in a 77% inhibition of tumor growth. Pharmacodynamic evidence of BRAF inhibition by ARQ 736 was demonstrated by a reduction in p-ERK staining of tumors via immunohistochemistry. Plasma from the ARQ 736 treated mice showed a reduction in human angiogenesis and cytokine related proteins relative to the control group. These include GM-SCF, Serpin E1, sICAM, and IL-8. The markedly reduced plasma IL-8 levels in response to ARQ 736 treatment detected by the antibody array were subsequently confirmed with a quantitative ELISA for human IL-8 (6-8 fold reduction). These results provide a basis for future clinical biomarker studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2517.
Abstract 2531: The selective CCR9 antagonist CCX8037 inhibits CCL25-CCR9-STAT3 mediated proliferation of pancreatic cancer cells
Authors: Lu Jianming , Shen Xiaoming , Mailey Brian , Lowy Andrew M. , Kim Joseph
Published: February 2011

Introduction: We previously reported the expression of CCR9 in pancreatic intraepithelial neoplasia (PanIN) and invasive pancreatic cancer tissues. Here, our objective was to investigate signaling downstream of CCR9 and to determine the anti-proliferative effects of the candidate therapeutic CCR9 antagonist CCX8037.

Methods: PanIN and corresponding invasive pancreatic cancer (5143PDA) and liver metastasis (5143LM) cell lines were derived from Pdx-1-Cre;LSL-K-rasG12D and Pdx-1-Cre;LSL-K-rasG12D/p53R172H mice. In culture, murine and human pancreatic cancer cells (PANC-1) were exposed to CCL25, the specific CCR9 ligand. The phosphorylation of STAT3, an established regulator of pancreatic cancer proliferation, was measured by Western blot assay and proliferation assays were performed with the small molecule CCX8037 and the JAK/STAT inhibitor AG490.

Results: First, we verified that exposure to CCL25 enhanced cell proliferation (20%-40%) in both murine and human pancreatic cancer cells. To investigate CCL25/CCR9 signaling, we exposed cells to CCL25 and observed increased phosphorylation of STAT3 in the cells. Blockade of STAT3 signaling with AG490 abrogated CCL25/CCR9-mediated proliferation in both murine and human pancreatic cancer cells. Pre-treatment of PanIN and pancreatic cancer cells with CCX8037 blocked CCL25-mediated phosphorylation of STAT3 and inhibited CCL25-mediated cell proliferation.

Conclusion: Our study demonstrates for the first time that the CCL25/CCR9 axis activates downstream STAT3 signaling to mediate proliferation in murine and human PanIN and pancreatic cancer cells. Additionally, our data shows that the candidate therapeutic agent CCX8037 effectively blocks CCL25/CCR9-mediated proliferation in pancreatic cancer and warrants investigation in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2531.
Abstract 2534: Suppression of prostate cancer growth by the Src signaling inhibitor, KXO1 (KX2-391)
Authors: Bu Yahao , Gao Lingqiu , Hangauer David , Gelman Irwin H.
Published: February 2011

KXO1 (KX2-391), developed as a Src tyrosine kinase signaling inhibitor, exhibits anti-proliferative activity against broad range of human cancer types in vitro, and has shown low toxicity in completed Phase I trials. We tested whether human and mouse prostate cancer (CaP) cell lines, which display activated levels of Src-family tyrosine kinases such as Src, Fyn and Lyn, could be inhibited by KXO1 compared to the broad tyrosine kinase inhibitor, Dasatinib (BMS-354825). Low nanomolar concentrations (9-75 nM) of KXO1 potently inhibited the proliferation of human CaP cell lines LNCaP, LNCaP-C4-2, PC3-LN4, LAPC-4 and DU145, and the CaP cell line, C2H, isolated from a castration-recurrent lesion in TRAMP mice. In contrast, whereas the GI50 of Dasatinib was equal to KXO1 for PC3-LN4 cells (9 nM), the GI50 for Dasatinib in LNCaP and DU145 cells was 10- and 2.7-fold higher than for KXO1, and Dasatinib failed to inhibit the proliferation of LNCaP-C4-2 cells at 10 μM. KXO1 exhibited a dose-dependent ability to inhibit anchorage-independent growth (AIG) of PC3-LN4, LNCaP and LNCaP-C4-2 cells. In contrast, Dasatinib caused less than 15% inhibition of AIG (compared to vehicle controls) in these CaP lines. Cell death, as measured by DNA fragmentation (Promega Cell Death ELISA kit), was induced after 24 h of KXO1 treatment (2-10X the GI50) of LNCaP and PC3-LN4 cells, but not induced by Dasatinib at 10X GI50 levels. We then tested the efficacy of KXO1 (vs. vehicle) in immunocompetent mice (C57BL/6) bearing small (∼150 mm3) syngeneic TRAMP C2H tumors using several treatment regimens that delivered 10 mg/kg/mouse/day. Mice receiving constant oral dosing (drinking water containing KXO1 in 5% sucrose) showed a 35-40% decrease in tumor growth rate, whereas mice dosed by oral gavage once or twice daily showed initial tumor growths followed by regression. Data will be shown comparing the efficacy of KXO1 against C2H tumors in immunocompetent versus SCID mice. Thus, KXO1 shows anti-CaP activity n vitro and in vivo irrespective of p53, AR or androgen-responsive status. Taken together, these data demonstrate strong preclinical evidence for the treatment of androgen-responsive and castration-recurrent CaP with KXO1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2534.
Abstract 2544: Analysis of the mechanism for acquiring resistance to 5-FU in gastric cancer cell lines
Authors: Nakamura Ayako , Hayashi Kazuhiko , Nakajima Go , Kuramochi Hidekazu , Okuyama Ryuji , Kamikozuru Hirotaka , Yamamoto Masakazu
Published: February 2011

Background 5-Fluorouracil (5-FU) still remains the key drug for the treatment of gastric cancer. Thus, analysis of the mechanism for acquiring resistance of gastric cancer cells to 5-FU and the development of effective chemotherapy for 5-FU-resistant gastric cancer are important.

Methods From the parent human gastric cancer cell lines (MKN45, MKN74, KATOIII and NCI-N87), we established several 5-FU-resistant cell lines (MKN45/5FU, MKN74/5FU, KATOIII/5FU and NCI-N87/5FU) by continuous exposure of the cells to progressively increasing concentrations of 5-FU over a year. We then compared the sensitivity of the parent cell lines to 5-FU-resistant cell lines by MTT assay. We also quantitatively evaluated the mRNA expression levels of four genes known to be associated with 5-FU metabolism, namely, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyltransferase (OPRT), by real-time RT-PCR assay.

Results The IC50 ratio of each 5-FU resistant cell line to its parent cell line was as follows; MKN45/5FU: 10.0; MKN74/5FU: 5.5; KATOIII/5FU: 2.2; NCI-N87/5FU: 4.8. Comparing with the corresponding parent cell lines, the 5-FU resistant cell lines, MKN45/5FU, MKN74/5FU, KATOIII/5FU and NCI-N87/5FU, showed a 0.62, 0.40, 0.37 and 0.56-fold decrease in TS gene expression, respectively. In addition, three of the four resistant cell lines showed a tendency towards increase in the gene expression level of DPD and decrease in that of TP. No significant changes in the expression level of the gene encoding OPRT was noted in any of the 5-FU-resistant cell lines.

Conclusion We established four 5-FU-resistant lines from the corresponding parent gastric cancer cell lines and demonstrated the various changes in the expression profiles of the genes known to be associated with 5-FU metabolism. These results suggest that the mechanism of acquiring resistance to 5-FU may vary among gastric tumors. To identify the genetic characteristics of these resistant cell lines, we propose to analyze the gene expression profiles at several time-points from the beginning of establishment of 5-FU-resistance, using microarray-based technology.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2544.
Abstract 2554: Copper transporter 2 (CTR2) is able to transport cisplatin and/or oxaliplatin in drug-resistant cell lines
Authors: Komlodi-Pasztor Edina M. , Murphy Robert , Wilkerson Julia , Fojo Tito
Published: February 2011

The clinical use of platinum (Pt) agents is often compromised by the occurrence of drug resistance. Studies have shown reduced intracellular Pt accumulation in cells resistant to Pt drugs. Copper transporters have been reported to have a role in Pt influx and efflux. While the first member of this family, CTR1, has been shown to transport Pt, the role of the structurally similar CTR2 in Pt transport is unclear. The aim of this study was to assess the role of CTR2 in Pt based drug-transport using KB-3.1 cervical carcinoma cells and its cisplatin (KB-CP20) or oxaliplatin (KB-OX60) resistant subclones. In previous studies we reported that compared to parental cells the resistant cell lines have lower levels of intracellular Pt following cisplatin (CIS) or oxaliplatin (OX) exposure. We transfected parental KB-3.1 cells and its drug resistant sublines with CTR1, CTR2 or both transporters, and incubated cells with either CIS or OX. Intracellular Pt accumulation was not increased in any of the transfected parental KB-3.1 cells. In contrast, KB-CP20 cells transfected with CTR1, CTR2 or both trasnporters accumulated more CIS but not more OX compared to non-transfected KB-CP20 cells; while transfected KB-OX60 cells accumulated higher levels of both CIS and OX compared to non-transfected KB-OX 60 cells. In both resistant cell lines, the increased intracellular Pt observed after transtfection of the transporters was accompanied by increased DNA platination. Given the clinical utility of OX in colon carcinoma we next determined intracellular CIS and OX levels, the expression of proteins involved in copper transport (ATP7A, ATP7B, CTR1 and CTR2), and the IC50 for CIS and OX of six colon carcinoma cell lines. We found an inverse correlation between intracellular Pt levels and the IC50 of both drugs, and correlations between: (1) the IC50 of CIS and expression levels of CTR1, (2) intracellular CIS levels and expression levels of ATP7A, (3) expression levels of ATP7B and CTR2, and (4) OX accumulation and expression levels of ATP7A. In summary, we have shown CTR2 is able to transport Pt-containing drugs and increases intracellular Pt accumulation that is accompanied by greater DNA-platination. In unselected colon cancer cells, we have shown that the IC50 of a Pt based drug is determined by the intracellular accumulation of that drug, and to varying extent by the expression of copper transporters.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2554.
Abstract 2557: GSTP1 is a determinant of temozolomiode resistance in human glioblastoma cells
Authors: Singh Simendra , Ali-Osman Francis
Published: February 2011

Temozolomide (TMZ) is a methylating agent used in first-line treatment of malignant gliomas and melanomas. Unfortunately, its use is limited by multifaceted resistance mechanisms. Although, the glutathione S-transferase P1 (GSTP1) protein metabolizes several alkylating and free radical generating anticancer agents, its role in TMZ metabolism and/or tumor resistance to TMZ are not known. In this study, we investigated the potential role of GSTP1-based metabolic detoxification of TMZ in glioma resistance to TMZ. The ability of GSTP1 to metabolize TMZ was assessed indirectly by measuring changes in the level of residual (free, un-metabolized) TMZ in a cell-free system containing TMZ, reduced glutathione (GSH) and human placental GSTP1 protein. We observed a time- and both GSH and GSTP1 concentration-dependent decrease in residual TMZ levels in reaction mixtures containing GSTP1 and after a 6 hr incubation, the level of free TMZ in GSTP1-containing reaction mixtures was decreased to 40% of controls without GSTP1. Presence of GSH alone resulted in only a 10% decrease in residual TMZ. Using three GSTP1 expressing glioma cell lines, viz, MGR1, MGR3 and U87, we examined the effect of siRNA-mediated knockdown of GSTP1 expression on tumor survival to TMZ and the level of TMZ-induced apoptosis using caspase-3/7 activation as endpoint. The TMZ IC50 values for MGR1, MGR3 and U87 of 1.31, 0.91 and 1.06 mM, respectively, were reduced significantly to 0.78, 0.6 and 0.69 mM at 48hrs following GSTP1 knockdown. Similarly, siRNA-mediated knockdown of GSTP1 increased TMZ-induced caspase-3/7 activation, significantly, in all three cell lines when exposed to TMZ for 48 hrs. Experiments evaluating the effect of combined knockdown of GSTP1 and O6-methylguanine-DNA methyltransferase (MGMT) on cellular sensitivity of TMZ suggest synergism between the two strategies. Overall, these results provide support for a pivotal role that GSTP1 may play in glioma cell resistance to TMZ-based chemotherapy and the potential of targeting GSTP1 as part of strategies to reverse this resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2557.
Abstract 256: Distinct roles for gain and loss of function p53 mutations in medullary thyroid cancer
Authors: Akeno Nagako , Ma Xiaolan , Wikenheiser-Brokamp Kathryn A.
Published: February 2011

Medullary thyroid carcinoma (MTC) accounts for 5-10% of all thyroid cancers and presents as metastatic diseases in ∼50% of patients. Patients with metastatic MTC are typically incurable because of the poor tumor response to current chemotherapeutics and radiation treatment. MTC occurs as sporadic tumors in ∼75% of cases. The remaining cases are familial and associated with either multiple neuroendocrine neoplasia syndromes or familial MTC syndrome. The Rearranged during Transfection (RET) proto-oncogene plays a key role in MTC development: however, the absence of RET mutations in ∼50% of sporadic cases and the variable clinical manifestations in patients with the same RET mutation provide compelling evidence that other genetic alterations are involved in MTC pathogenesis. Recent studies in human tumors and mouse models suggest that the Rb and p53 pathways cooperate to suppress MTC evolution. Although some p53 mutations cause a loss of function, most p53 mutations detected in human cancers result in expression of a mutant p53 protein which may exhibit gain of function properties. The objective of this study was to identify the function of p53 mutant proteins commonly expressed in human cancers in MTC pathogenesis. A conditional mouse model was generated in which MTC is initiated by Rb inactivation. Using this model, the consequences of p53 mutant protein expression on MTC initiation and progression were compared to p53 deletion. Loss of a single copy of p53 led to accelerated medullary thyroid tumor progression resulting in decreased survival. Loss of both p53 alleles resulted in a markedly more severe phenotype leading to a median survival of 4 months. Expression of the structural p53R172H mutant, but not the DNA contact p53R270H mutant, resulted in accelerated death compared with p53 deletion demonstrating that p53R172H has a gain of function property. Interestingly, mice heterozygous for p53R172H/+ and p53R270H/+ showed similar survival as mice with loss of a single p53 allele providing evidence that neither p53 mutant protein functions in a dominant negative fashion to promote MTC initiated by Rb loss. These results clearly demonstrate that p53 suppresses the progression of MTC initiated by Rb loss and indicate that gain of function p53 mutations confer poorer prognosis than p53 loss. Given the critical roles played by the Rb and p53 pathways in tumor response to therapy, identifying key Rb and p53 functions in MTC pathogenesis is important for future design of therapies to combat these currently treatment resistant malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 256.
Abstract 2566: Mesenchymal to epithelial transition associates with drug resistance in tongue squamous cell carcinoma cell line
Authors: Liu Xiqiang , Jiang Lu , Shi Fei , Zhou Xiaofeng
Published: February 2011

Epithelial to mesenchymal transition (EMT) has been implicated in altered drug sensitivity to various chemotherapeutic agents. Herein, we investigate whether this phenomenon would play a role in acquired resistance to cisplatin. We firstly established a cisplatin-resistant subline (UMcis, IC50 = 19.8 µM), which was derived from the cisplatin-sensitive tongue squamous cell carcinoma cell line, UM1 (IC50 = 3.6 µM). The UMcis cells showed cross-resistance against other chemotherapeutic drugs with divergent mechanisms of action [5-fluoro-uracil (5-FU), paclitaxel and epidermal growth factor receptor-tyrosine kinase inhibitor erlotinib]. Genome-wide expressional profiling was performed on the paired cell lines. Interestingly, a large number of genes associated with EMT (or reversed EMT, MET) were differentially expressed between the two cell lines. We confirmed that increased expression of E-Cadherin combined with the loss of Vimentin, Slug, Twist1 and MMP2 by immunoblotting was associated with drug resistance. Importantly, downregulation of E-Cadherin in UMcis cells not only increased the expression of mesenchymal markers, such as Vimentin, Slug and Twist1, but also restored drug sensitivity. Moreover, concurrent treatment with transforming growth factor-beta 1 and epidermal growth factor induced EMT in the UMcis cells, which also led to altered chemo-responsiveness. Therefore, our results suggest that induction of MET may contribute to the decreased efficiency of cisplatin-based chemotherapy and acquired drug resistance in tongue cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2566.
Abstract 257: R4 knock-out mice showed lung hyperplasia and susceptible to urethane-induced lung carcinogenesis
Authors: Choi Ji Won , Kim Il Yong , Shin Jae Hoon , Lee Seo Hyun , Bae Yun Soo , Seong Je Kyung
Published: February 2011

Lung cancer is responsible for 1.3 million deaths worldwide annually. There have been several reports on candidate genes driving lung cancers including ras. However still it is needed to find new genes which can cause lung cancers.

Recently we developed R4 knock-out (KO) mice which showed larger relative lung weight and volume than age-matched wild-type (WT) mice. Lung hyperplasia was found with increased alveolar thickness in 6, 18 and 40 week-old R4 KO mice. Also, this finding was observed in E16.5 and 18.5 day KO mice. Cell cycle markers including PCNA and cyclinD1 were highly expressed in the lung of R4 KO mice than WT mice. PCNA positive cells were mostly type 2 pneumocytes.

To determine the susceptibility of lung cancer in R4 KO mice, we induced lung cancer by the injection of 1g/kg urethane weekly for a total of eight doses. Twenty weeks after the initial injection, mice were sacrificed, and the lungs were fixed, and the number of tumors on the lung surface was assessed. Also lung tumors were scanned by micro-CT at the end of the lung carcinogenesis protocol for measure the tumor diameter. Surface lung tumor number is significantly increased in KO mice. And tumor size is also significantly larger in KO mice than WT. Histological findings showed more severe well differentiated lung adenoma in urethane-injected R4 KO mice.

We conclude that R4 KO mice is susceptible to tumorigenesis in the urethane model and the functional loss of R4 gene may cause lung cancer. Therefore, R4 gene has a possibility as a potential target for chemoprevention of lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 257.
Abstract 2572: Quantitative proteomic and genomic analyses reveal that stress hormones induce a drug resistant phenotype of triple negative breast cancer cells treated with paclitaxel
Authors: Flint Melanie S. , Hood Brian L. , Bateman Nicholas W. , Conrads Thomas P.
Published: February 2011

Triple negative breast cancer (TNBC) is clinically characterized as an aggressive form of cancer which is unresponsive to hormone and trastuzumab based therapies and associated with poorer overall patient prognosis. TNBC can be treated with chemotherapeutic agents but unfortunately typically half of patients do not respond to treatment because their breast cancer cells become resistant. Recent evidence suggests that psychological distress in cancer patients equates to poor chemotherapeutic response, indeed the National Cancer Institute reports that 50% of cancer patients report anxiety due to the cancer diagnosis, the fear of dying and chemotherapy. Historically, single markers depicting breast cancer prognosis have failed to readily translate to the clinic therefore the expression pattern of multiple molecules may prove better predictors of drug resistance to chemotherapeutic agents. We hypothesize that drug resistance in breast cancer may arise in part due to stress hormone-induced alterations in intrinsic cellular proteins/genes in cancer cells. To examine the effects of stress, via stress hormones on drug resistance MDA-MB-231 breast cancer cells were incubated with paclitaxel in the presence of physiological concentrations of cortisol, norepinephrine and epinephrine for 48 h. We utilized a quantitative proteomic strategy, Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), and LC-MS/MS to compare differences in protein abundance between paclitaxel-treated and stress hormone/paclitaxel-treated cells. For proteomic profiling, paclitaxel-treated cells cultured in unlabeled (no stress hormones) and labeled (stress hormones) SILAC media were mixed, lysed, and proteins were resolved by 1D-SDS PAGE, digested in-gel with trypsin and analyzed by LC-MS/MS. For genomic analyses, total RNA was isolated using TRIzol, cRNA was generated and hybridized to Affymetrix GeneChip HGU133 plus 2.0 arrays. We identified 220 proteins and 1201 genes whose expression levels were significantly altered by stress hormones. We further identified a unique molecular profile of predictive candidate biomarkers related to stress hormone- induced drug resistance including a significant decrease in the mitotic checkpoint serine/threonine-protein kinase BUB1 and the DNA damage response enzyme PARP-1, and increases in the immunophillin, FKBP-5 and the cell proliferation/apoptosis regulator C-MYC. In conclusion, our results demonstrate that stress hormones promote resistance to paclitaxel through modulation of several markers of drug resistance in TNBC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2572.
Abstract 2575: Activation of antibody drug conjugate MDX-1203 by human carboxylesterase
Authors: Derwin Dan , Passmore David , Sung Janette , Tengco Daniel , Lee Brian , Aguilar Barbara , Chen Tim , Zhang Allen , Sufi Bilal , Cong Charlie , Salles Adam , Huber Mary , Stevens Alice , Rao Chetana , Gangwar Sanjeev , Deshpande Shrikant , Rangan Vangipuram S.
Published: February 2011

MDX-1203 is a human anti-CD70 antibody conjugated to a DNA alkylating cytotoxic Drug A. Drug A is an enzyme-activated prodrug that requires cleavage of its peptide linker and removal of an ester-linked protecting group for cytotoxic activity. Upon internalization of MDX-1203 in targeted cancer cells, peptide linker in drug A is cleaved by lysosomal enzymes to produce Drug B; which is further processed by intracellular enzymes to produce active drug. The purpose of this study was to determine if Drug B and n-acetyl-cysteine-conjugated Drug A (NAC-Drug A) are substrates of recombinant human carboxylesterase, and to determine kinetic parameters for the enzyme iso-forms rhCE1 and rhCE2. Furthermore, a comparison of rhCE1 and rhCE2 kinetics was made with CPT-11, a commercially available prodrug used to treat colon cancer that is preferentially activated by human CE2. Human carboxylesterase 1 and 2 with a C-terminal histidine tag were cloned and expressed in CHO cells. The enzymes were purified using immobilized metal ion affinity chromatography (IMAC), and their identities were confirmed by N-terminal protein sequencing and Western blot using an anti-his antibody. Enzyme reactions were carried out at 37°C by incubation with Drug A, NAC-Drug B, or CPT-11. The amount of enzymatically released drug product was determined by RP-HPLC. Drug B was determined to be a substrate for both rhCE1 and rhCE2; however, its Km was much lower with rhCE2 (1.5 uM) as compared to rhCE1 (53 uM), and the overall enzyme efficiency (Vmax/Km) was approximately 50 fold higher for rhCE2 as compared to rhCE1. Similarly, Drug B and NAC-Drug A were both found to be better substrates for rhCE2 as compared to rhCE1. Furthermore, recombinant human CE2 cleaves ester group in NAC-Drug A, 20 fold more efficiently as compared to CPT-11. These results indicate that expression of carboxylesterase 2 in targeted cancer cells is probably important for intracellular activation of MDX-1203.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2575.