bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1538: Role of mitochondrial dysfunction in GPX regulation and glutathione metabolism
Authors: Lu Weiqin , Feng Li , Zhang Hui , Huang Peng
Published: February 2011

Many cancer cells exhibit mitochondrial respiratory dysfunction, which is associated with metabolic alterations, change in cellular redox states, and drug resistance. It is also known that elevated glutathione in cancer cells may promote their survival and resistance to anticancer agents. However, the exact relationship between mitochondrial dysfunction and alterations in cell redox states and viability remain elusive, and the underlying regulatory mechanisms are unclear. In this study, we used a dominant negative form of mitochondrial DNA polymerase γ (POLGdn) expressed in a T-Rex293 tet/on system as a model to induce mitochondrial dysfunction, and showed that suppression of mitochondrial respiration caused a significant increase in ROS generation. Mitochondrial dysfunction also led to a significant increase in glutathione, along with an elevated expression of its rate-limiting enzyme GCLC and the upstream transcription factor Nrf-2. Interestingly, the mRNA expression of glutathione peroxidases GPX1 and GPX4 remain the same, while the GPX proteins and enzyme activity were significantly increased in the mitochondrial defective cells. Since GPX1 and GPX4 are selenium-containing enzymes, they require selenocysteine-tRNA for their protein synthesis. We further demonstrated that selenocysteine-tRNA expression and its transcription factor ZNF143 protein were significantly increased after induction of mitochondrial dysfunction. Furthermore, siRNA knockdown of ZNF143 led to a decrease of GPX activity, an increase in ROS, and a reduction in cell viability in the mitochondrial defective cells. Similarly, a knockdown of GPX1 by shRNA in the mitochondrial defective cells also compromised cell survival. We also observed that ZNF143 protein level was decreased in the GPX1-knockdown cells, suggesting that GPX1 has a positive feedback role in regulating ZNF143 expression. These data suggest that mitochondrial dysfunction led to an activation of glutathione synthesis and an increase in GPX activity through upregulation of the transcription factors Nrf-2 and ZNF143, respectively, and that mitochondrial dysfunction in cancer cells seems to set a new balance between the increase in ROS generation and the elevation in glutathione antioxidant system to maintain cell survival. As such, targeted inhibition of selenocysteine-tRNA, selenoenzymes, or glutathione synthesis may be potential therapeutic strategies for cancer treatment.

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 1538.
Abstract 1540: MAPK signaling profiles differ between 1,25D-sensitive HL60G and 1,25D-resistant 40AF human leukemia cells
Authors: Chen-Deutsch Xiangwen , Studzinski George P.
Published: February 2011

The resistance to 1,25-dihydroxyvitamin D3 (1,25D) could be an obstacle for potential clinical use of vitamin D in differentiation therapy of leukemia. 40AF cells are a subline of HL60 cells and provide a model for studies of 1,25D-resistance. It is known that 40AF cells grow more rapidly than the parental cells, and have a high percentage of cells in S phase. Also, the addition of the plant antioxidant carnosic acid and a kinase inhibitor SB202190 (the combination dubbed DCS) can partially overcome the resistance to 1,25D. To better understand the basis for this resistance we examined MAPK network profiles, previously shown to be important for HL60 cell differentiation, on mRNA level by RT2-PCR arrays. The results showed that 17 out of 84 genes studied had significantly altered mRNA levels (more than 2 fold) in 40AF cells compared to HL60G cells. Eighteen genes had lower basal level of expression in 40AF compared to HL60G cells, and after partial reversal of resistance by DCS treatment these genes showed increased expression; in contrast, 20 genes had higher basal levels in 40AF cells and their expression was decreased by DCS, suggesting that these genes may be involved in 1,25D resistance. Also, DCS treatment of 40AF cells changed the expression of the RAS-ERK pathway components more obviously than the JNK and the p38MAPK pathways. Mitotic cyclins and CDKs had a generally enhanced expression in 40AF cells compared to HL60G, and their expression decreased following DCS treatment, consistent with the slow down of the cell cycle by DCS, perhaps also because several CKIs, including p21Cip1, dramatically increased. Studies using Phospho-MAPK arrays showed that treatment of both HL60G and 40AF cells with 1,25D or DCS altered the levels of phosphorylated proteins of MAPK family members P-ERK2 in the ERK pathway, P-JNK2 in the JNK pathway, P-p38 alpha/delta/gamma in the p38 pathway, P-Akt1 in the PI3/Akt pathway. The gene expression studies will be validated by Westerns, and MAPK activity by kinase assays. In summary, MAPK signaling networks may play important roles in 1,25D resistance in AML cells, and modification of network expression can influence cell cycle progression and cell differentiation to partially reverse the resistance to 1,25D of non-responding leukemia cells.

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 1540.
Abstract 1541: Role of GSH in mediating stromal-leukemia interaction and promoting cell survival and drug resistance in chronic lymphocytic leukemia
Authors: Zhang Wan , Trachootham Dunyaporn , Liu Jinyun , Keating Michael J. , Huang Peng
Published: February 2011

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The interaction between CLL cells and the bone marrow stromal environment is thought to play a major role in promoting the leukemia cell survival and drug resistance. Here we show a novel biochemical mechanism by which the bone marrow stromal cells exert a profound influence on the redox status of primary CLL cells and enhance their ability to sustain oxidative stress and drug treatment. Fresh leukemia cells isolated from the peripheral blood of CLL patients exhibited two major redox alterations when they were cultured alone: a significant decrease in cellular glutathione (GSH) and an increase in basal ROS levels. However, when cultured in the presence of bone marrow stromal cells, CLL cells restored their redox balance with an increased synthesis of GSH, a decrease in spontaneous apoptosis, and an improved cell survival. Further study showed that CLL cells were under intrinsic ROS stress and highly dependent on GSH for survival, and that the bone marrow stromal cells promoted GSH synthesis in CLL cells through a novel biochemical mechanism. Cysteine is a limiting substrate for GSH synthesis and is chemically unstable. Cells normally obtain cysteine by uptaking the more stable and abundant precursor cystine from the tissue environment and convert it to cysteine intracellularly. We showed that CLL cells had limited ability to take up extracellular cystine for GSH synthesis due to their low expression of the transporter Xc-, but had normal ability to uptake cysteine. In the co-culture system, the bone marrow stromal cells effectively took up cystine and reduced it to cysteine for secretion into the tissue microenvironment to be taken up by CLL cells for GSH synthesis. The elevated GSH in CLL cells in the presence of bone marrow stromal cells significantly protected the leukemia cells from stress-induced apoptosis, and rendered them resistant to standard therapeutic agents such as fludarabine and oxaliplatin. Importantly, disabling of this protective mechanism by depletion of cellular GSH using a pharmacological approach potently sensitized CLL cells to drug treatment, and effectively enhanced the cytotoxic action of fludarabine and oxaliplatin against CLL in the presence of stromal cells. This study reveals a key biochemical mechanism of leukemia-stromal cells interaction, and identifies a new therapeutic strategy to overcome drug resistance 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 1541.
Abstract 1542: Molecular mechanisms associated with sensitivity/resistance to PM02734 (Irvalec®), a novel marine-derived cyclic depsipeptide
Authors: Serova Maria , Bieche Ivan , Vidaud Michel , Aracil Miguel , Faivre Sandrine , Raymond Eric
Published: February 2011

Background: PM02734 (Irvalec®) is a novel synthetic depsipeptide derived from kahalalide F currently in clinical trials with evidence of antitumor activity. This study aimed to evaluate the mechanisms yielding antiproliferative effects of PM02734 in human cancer cells.

Materials and Methods: Antiproliferative effects of Irvalec used MTT assay. Baseline and phosphorylated protein levels were assessed using Western blot. Relative level of mRNA expression was evaluated using quantitative qRT-PCR.

Results: PM02734 displayed antiproliferative effects in a panel of 20 cancer cell lines with IC50s ranging 0.4-9µM in breast (ZR-75-1, SKBR3 and MDA-MB-361) and colon (Colo205 and HCC2998) cancer cell lines. Unspecific changes were observed in MAPK and AKT intracellular signaling under exposure to PM02734 despite a decreased Erb-B3 mRNA expression and Erb-B3 phosphorylation at 24h. To study mechanisms associated with resistance to PM02734, we developed DU-PM cells using stepwise exposures to increasing concentrations of PM02734 in DU145. After >6 months exposure to PM02734, DU-PM cells were 4-fold more resistant to PM02734 than parental DU145 cells. Resistance was associated with decreased mRNA expressions of Erb-B3/Erb-B4 and vimentin. Conversely, the mRNA expressions of anti-apoptotic genes such as CDKN1A/p21 and BCL2 were significantly increased in PM02734-resistant cells. Interestingly, DU-PM cells were more sensitive to EGFR inhibitors such as erlotinib, gefitinib and lapatinib than parental DU145 cells. These results provide a rational basis for for further exploring PM02734 in combination with EGFR inhibitors in solid tumors.

Conclusions: PM02734 displays antiproliferative effects against a large variety of cancer cell lines at pharmacological concentrations. Acquired resistance to PM02734 associated with low Erb-B3 expression that may be regarded as predictive factor of sensitivity. The cells with acquired resistance to PM02734 display increased sensitivity to EGFR inhibitors.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 1542.
Abstract 1558: STX140 but not paclitaxel inhibits mammary tumor initiation, progression and metastasis in C3(1)/SV40 T/t-antigen transgenic mice
Authors: Foster Paul A. , Newman Simon P. , Day Joanna M. , Leese Matthew P. , Potter Barry VL , Reed Michael J. , Purohit Atul
Published: February 2011

New chemotherapeutic options for late-stage metastatic breast cancer are urgently required. Currently, the taxanes are considered one of the most effective treatments for late-stage cancer treatment.

Paclitaxel and the novel cytotoxic agent STX140, compounds that have shown significant activity against numerous xenograft mouse models of cancers, are here investigated in the C3(1)/SV40 T/t-antigen mouse model of spontaneous breast cancer. This mouse model recapitulates important histopathological and molecular alterations in mammary cancer development over a highly predictable time-course. At 8 weeks of age, the female animals develop low-grade mammary intra-epithelial neoplasia (MIN) lesions. By 12 to 14 weeks of age this progresses to high-grade MIN, which is similar to human ductal carcinoma in situ. From 15 weeks of age onwards invasive, metastatic carcinomas are observed. STX140 and paclitaxel were administered to these mice at 12 weeks of age (early intervention) and when tumors had reached 0.5 cm (late intervention).

Results demonstrated that all mice in the control and paclitaxel treated groups at early intervention developed palpable mammary cancer which grew to 3112 mm3 by 25 weeks of age and led to the termination of these animals at week at this time point. However, STX140, when administered at week 12 onwards, blocked disease progression and no palpable tumor develop in 47% of tumors and significantly inhibited the growth of tumors that did develop. This resulted in a significant (P < 0.01) survival advantage for animals treated with STX140. Conversely, in C3(1)/SV40 mice, paclitaxel failed to inhibit tumor growth and therefore did not confer a survival benefit. In the late treatment groups, STX140 significantly (P < 0.05) inhibited primary mammary cancer growth and also resulted in a significant (P < 0.001) survival advantage compared to control animals. Furthermore, it was observed that paclitaxel treatment increased liver metastases in these mice.

To conclude, STX140 has a greater anti-cancer efficacy and conferred an improved survival outcome compared to paclitaxel in a clinically relevant transgenic mouse model of metastatic breast 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 1558.
Abstract 1561: Guaiacol glyceryl ether, a novel agent to reduce MUC1 expression in breast cancer cells
Authors: Smith Joshua S. , Colon Jimmie , Madero-Visbal Rafael , Konduri Santhi , Baker Cheryl H.
Published: February 2011

Purpose: The overexpression and loss of polarity of Mucin1 (MUC1) has been documented in aggressive breast cancer. We have identified guaifenesin as a novel treatment to reduce or eliminate MUC1 expression on human breast cancer cells.

Methods: MCF-7 and ZR-75-1 human breast cancer cells were grown for 48 hours in varying concentrations of guaifenesin to determine if a dose dependent effect on MUC1 protein expression resulted with treatment. In addition, cells were also harvested, RNA isolated, reverse transcribed and PCR was performed using ABI quantitative real time PCR machine. In another set of experiments, MCF-7 cells were plated and grown for 72 hours in the absence or presence of 12.5 mM guaifenesin and the cells were harvested and stained with propidium iodine for analysis by flow cytometry. The Cell-Titer Glo assay was performed on MCF-7 and ZR-75-1 cells incubated for 48 hours in the presence of guaifenesin at varying concentrations and the LD50 was determined. In a final set of experiments, MCF-7 cells, incubated in the presence or absence of guaifenesin, were analyzed for their ability to migrate /proliferate in vitro by using a standard scratch assay. For in vivo studies ovarectomized female nude mice were injected with MCF-7-luciferase tagged cells into the mammary fat pad 24 hours after an estrogen pellet was implanted into the right flank. Treatment (vehicle or 400 mg/kg/daily) was initiated after 10 days and tumor growth was monitored through Kodak imaging system. Six weeks after the treatment tumors were isolated, measured and processed for immunohistochemistry to analyze the expression of MUC1, TUNEL and CD31.

Results: Initially, MUC1 gene expression was confirmed in MCF-7 and ZR-75-1 human breast cancer cell lines. Following confirmation, the LD50 was determined to be 12.5 mM. MUC1 gene expression was successfully decreased in a dose-dependent manner in both MCF-7 and ZR-75-1 cell lines when treated with guaifenesin. When MCF-7 cells are treated with 12.5mM guaifenesin, G1 arrest occurs within 24 hours and by 72 hours, 94.2% of cells are in G1 compared to 76.2% in control (drug free) conditions. In addition, there was a significant decrease in the ability of guaifenesin treated MCF-7 cells to migrate into the scratch compared to the control (untreated) cells. In vivo studies revealed that guaifenesin significantly decreased the breast tumor weight and volume which was associated with a low expression of MUC1 in tumor sections as evaluated by immunohistochemistry.

Conclusion: These findings suggest that guaifenesin may provide a novel approach to inhibit breast cancer growth.

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 1561.
Abstract 1563: Role of EGFR during the transition of inflammation to dysplasia in a colitis-associated colon cancer model
Authors: Pagan-Ortiz Beatriz , Isidro Angel A. , Coppola Domenico , Wu Jie , Appleyard Caroline B.
Published: February 2011

Background: The underlying mechanisms in ulcerative colitis-associated dysplasia are poorly understood. Neurokinin-1 receptors (NK-1R) have been shown to be involved in chronic colonic inflammation, and we have demonstrated their over-expression in patients with dysplasia. The NK-1R has been found to crosstalk with the epidermal growth factor receptor (EGFR). EGFR is related to the development of some cancers including colorectal cancer. Erlotinib, a potent inhibitor of the EGFR tyrosine kinase, has been shown to inhibit the intracellular EGFR signaling pathway and to block non-small cell lung cancer and pancreatic tumor growth; however its role in the genesis of dysplasia is unknown. Aim: The aim of this study was to investigate the role of EGFR in a novel model of colitis-associated dysplasia using a small molecule inhibitor, Erlotinib. Methods: Sprague Dawley rats received trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol, ic.), followed six weeks later by reactivation with TNBS (5 mg/kg, iv.) for three days. To induce colitis-associated dysplasia, the rats then received TNBS (iv.) twice a week for ten weeks. One group received the Erlotinib (10 mg/kg, ip.) for one week before the start of the reactivation of the colitis and two weeks after (21 days); the rest received the vehicle. After sacrifice, the colons were removed and analyzed for damage and expression of NK-1R, Cox-2, EGFR, and the downstream signaling components. Results: Animals receiving Erlotinib had no significant difference in macroscopic or microscopic damage from controls. Pathological analysis however revealed a decrease of the incidence of dysplasia in animals treated with Erlotinib when compared with the vehicle-treated animals. This decrease was most evident in both the mid and the distal colon (15% vs. 53% and 20% vs. 61%). Colonic tissues from Erlotinib-treated animals also had decreased expression of NK-1R, Cox-2, pErk ½, and Akt compared to the vehicle-treated group. Conclusion: Our results suggest that Erlotinib can delay the development of colitis associated dysplasia, suggesting a potential therapeutic use for Erlotinib in patients with long-standing colitis. Supported in part by 1U56 CA126379-01 and 1F31 GM078951.

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 1563.
Abstract 1571: Nek2 as an effective molecular target for cancer treatment
Authors: Kokuryo Toshio , Senga Takeshi , Yokoyama Yukihiro , Nagino Masato , Hamaguchi Michinari
Published: February 2011

Nek2 (NIMA-related kinase 2) is a member of the serine/threonine kinase family, Nek, structurally related to the essential mitotic regulator NIMA. Nek2 is involved in cell division and mitotic regulation by centrosome splitting. Increased expression of Nek2 has been reported in cell lines derived from cervical and prostate cancer as well as lymphomas. We, therefore, examined the expression levels of Nek2 in various cancer cell lines, such as cholangiocarcinoma (HuCCT1, TFK1, HuH28, CCKS1, ETK1, RBE, IHGGK), breast cancer (BT20, BT474, Hs578T, MCF7, MDA-MB-231, T47D, ZR-75-1) and colorectal cancer (HCT-15, DLD-1, Colo205, Colo320). These cancer cells expressed higher levels of Nek2 mRNA and protein than those of HFF, a cell line derived from normal human fibroblast. The expression of Nek2 was also elevated in breast cancer and colorectal cancer tissue in compared with that of normal tissue. The Nek2 suppression with siRNA resulted in the inhibition of cell proliferation and induced cell death in these cancer cell lines. We next examined the effect of Nek2 siRNA treatment on the growth of cholangiocarcinoma tumor in xenograft-nude mouse model. Mean tumor volumes in Nek2 siRNA-treated mice were substantially reduced in comparison with those of the control mice. The Nek2 expression in the tumor tissues was dramatically suppressed in Nek2 siRNA treated tumors, as compared with those of control siRNA treated tumors. Nek2 siRNA also resulted in reduction of tumor size as compared with those of control siRNA injection in breast cancer tumor and colorectal cancer tumor. To further evaluate the effects of Nek2 siRNA treatment, we challenged siRNA in the peritoneal dissemination model. It should be noted that peritoneal dissemination is one of the major undesirable complications frequently associated with inoperable cases. In peritoneal dissemination model, Nek2 siRNA-treated mice showed statistically longer survival periods in comparison with those of the control siRNA-treated mice. Survival rates of the Nek2 siRNA-treated mice were approximately 1.5 times higher than those of the control siRNA-treated mice. Taken together, for the improvement of cancer therapy, identification of specific molecular targets critical for its tumorigenicity is an important issue. Nek2 plays a critical role in tumorigenic growth of various cancers, and inhibition of Nek2 expression with its siRNA causes suppression of cancer growth and survival. Our data in vivo provides a strong rationale for the further investigation of Nek2 inhibitors as a new cancer therapy, holding the potential to provide regression of multiple human 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 1571.
Abstract 1577: Contributions of KIT and ABL to the therapeutic responses of GIST and CML cells to imatinib mesylate
Authors: Brown Matthew F. , Duensing Stefan M. , Duensing Anette U.
Published: February 2011

Most GISTs are caused by activating mutations of the KIT receptor tyrosine kinase and can effectively be treated with the small molecule kinase inhibitor imatinib mesylate (IM). IM is a potent inhibitor of KIT as well as ABL, a kinase that is constitutively activated through a chromosomal translocation (BCR-ABL) in chronic myeloid leukemia (CML). Since both KIT and ABL are expressed in GIST as well as CML cells, the present study was designed to determine the individual contributions of KIT and ABL inhibition, respectively, to the therapeutic responses in both malignancies. As expected, siRNA-mediated knock-down of KIT in the human GIST cell line GIST882 led to a significant increase of apoptosis and reduced cellular proliferation as measured by TUNEL and luminescence-based assays. Depletion of ABL1 in human K562 CML cells led to similar results. Immunoblotting for PARP cleavage as well as p27(Kip1) and cyclin A corroborated these findings. Reduction of ABL1 protein levels in GIST cells, however, was only associated with a minimal increase of apoptosis. Nevertheless, it resulted in a significantly reduced cellular proliferation when compared to controls and had approximately 50% of the anti-proliferative effect detected after knock-down of KIT in GIST cells. Combined knock-down of ABL1 and KIT in GIST did not show an additive effect. In contrast, siRNA-mediated knock-down of KIT in K562 cells did not have any significant effects on either cell survival or proliferative activity. Combined depletion of ABL1 and KIT in CML cells had a similar effect as knock-down of ABL1 alone. Immunoblotting analysis confirmed respective siRNA knockdowns, inhibition of downstream pathways and cellular proliferation as well as induction of apoptosis. In conclusion, ABL inhibition is unlikely to contribute to the induction of apoptosis after IM-treatment of GIST cells, however, it may cooperate with KIT inhibition to inhibit GIST cell proliferation. In contrast, inhibition of KIT does not seem to play a role in the therapeutic effect of IM in CML cells. Our results have implications for future strategies to improve the therapeutic effectiveness of targeted therapies in GISTs.

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 1577.
Abstract 1581: IL-13 mediates signal transduction through IL-13 receptor α2 in pancreatic ductal adenocarcinoma: role of IL-13 pseudomonas exotoxin in pancreatic cancer therapy
Authors: Shimamura Takeshi , Fujisawa Toshio , Husain Syed R. , Joshi Bharat H. , Watanabe Seitaro , Kato Shingo , Kobayashi Noritoshi , Kubota Kensuke , Nakajima Atsushi , Puri Raj K.
Published: February 2011

Purpose: Interleukin-13 receptor alpha 2 (IL-13Rα2) is a tumor antigen, which is overexpressed in certain human tumors. However, its expression and significance in pancreatic cancer and whether IL-13 can signal through IL-13Rα2 in cancer is not known. In addition, it is not known whether IL-13-PE can target pancreatic cancer for therapy.

Experimental Design: The expression of IL-13Rα2 was assessed in pancreatic cancer samples by IHC; and in cell lines by flow cytometry and RT-PCR. The role of IL-13Rα2 was examined by IL-13 induced signaling in pancreatic cancer cell lines. IL-13Rα2 positive tumors were targeted by IL-13PE cytotoxin in vitro and in vivo in orthotopic murine model of human pancreatic cancer.

Results: Seventy one percent pancreatic tumor samples overexpressed moderate- to high-density IL-13Rα2 compared to normal pancreatic samples. IL-13 induced TGF-β1 promoter activity in IL-13Rα2 positive tumor cells and in cells engineered to express IL-13Rα2 but not in IL-13Rα2-negative or RNAi knockdown cells. c-Jun and c-Fos of AP-1 family nuclear factors were activated by IL-13 only in IL-13Rα2-positive cells. In orthotopic mouse model, IL13-PE significantly decreased tumor growth when assessed by whole body imaging. In addition, IL13-PE prolonged the mean survival time of treated animals. Similar results were observed in mice xenografted orthotopically with a surgically resected human pancreatic tumor sample.

Conclusions: These results indicate that IL-13Rα2 is a functional receptor as IL-13 mediates signaling in human pancreatic cancer cell lines. IL-13 causes TGF-β activation via AP-1 pathway, which may cause tumor induced immunosuppression in the host. In addition, IL13-PE cytotoxin may be an effective therapeutic agent for the treatment of pancreatic 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 1581.
Abstract 1587: Targeted deletion of Fra-1 blocks oncogenic K-ras induced lung tumor development and progression in mice
Authors: Vaz Michelle , Reddy Narsa M. , Wagner Erwin F. , Reddy Sekhar P.
Published: February 2011

Background: Fra-1 (Fosl1) is a dimeric partner of AP-1 transcription factor and regulates gene expression in a context-dependent (cell-type and stimulus-specific) manner through the TPA response element (AP-1 site). We have previously shown that Fra-1 is strongly activated in lung epithelial cells by cigarette smoke, a major determinant of lung cancer, as well as tumor promoting mitogenic and pro-inflammatory stimuli. Ectopic expression of Fra-1 promotes human lung cancer cell progression in vitro, and tumor development in nude mice. We hypothesized that Fra-1, an effector of ERK signaling, is required for oncogenic K-ras promoted lung tumor development in vivo. Methods: To test our hypothesis and to better understand the role of Fra-1 in lung tumor development and progression in vivo, we have generated transgenic mice bearing floxed allele Fra-1 (Fra-1FF) and LSL K-rasG12D by cross-breeding Fra1F/F and LSL K-rasG12D mice (NCI), which carry a latent point-mutant allele of K-ras (K-rasG12D). Fra1wt/wt +LSL-K-rasG12D mice (abbreviated as Kras+/−) and Fra1F/F+ LSL-K-rasG12D mice (Fra1FF- K-rasG12D) were instilled intratracheally with adenovirus Cre (2.5 ×107 pfu) and then were allowed to recover for an additional 13 weeks. Mice were sacrificed, lungs were fixed, and lung tumor induction was analyzed by histopathology. Results: As anticipated, activation of K-ras oncogene by Ade-Cre instillation induced lung tumor development in Kras+/− mice bearing Fra-1 wild type alleles. Hyperplastic lesions with papillary structures and adenomas were clearly visible in these mice. Strikingly, the lung tumor formation in Fra-1 floxed mice bearing LSL-K-rasG12D allele (Fra1F/F+ LSL-K-rasG12D mice) infected with Adeno-cre virus was markedly reduced. In all animals, with exception of few hyperplastic lesions, no adenomas were detected in mice lacking Fra-1. Conclusions: The present data suggest that Fra-1 is critical for oncogenic K-ras promoted lung tumor development 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 1587.
Abstract 1589: Low shear stress induces the novel tumor endothelial marker CLEC14A that mediates cell migration and vascular development
Authors: Mura Manuela , Zhuang Xiadong , Vorschmitt Henrik , Swain Rajeeb K. , Durant Sarah , Herbert John M. , Sheldon Helen , Andre Maud , Sanderson Sharon , Reynolds Gary , Glen Katie , Luu Nguyet-Thin , McGettrick Helen , Antczak Phillip , Falciani Francesco , Nash Gerard , Nagy Zsuzsanna , Bicknell Roy
Published: February 2011

Experimental evidence using animal models has proven the efficacy of targeting a tumor endothelial marker (TEM) in eradicating large solid tumours. It follows that a well characterised tumor endothelial marker would offer much promise as an anti-cancer target. Despite many efforts by numerous groups, a well defined tumor endothelial marker in man has remained elusive. CLEC14A is a novel endothelial specific C-type lectin that is a member of the endosialin/TEM1 and thrombomodulin family of lectins. Protein sequence alignment of CLEC14A shows conservation from fish to man and endothelial specificity of expression is seen in the embryonic zebrafish. Detailed analysis of CLEC14A expression using human tissue arrays showed that CLEC14A is absent in human adult tissue but highly expressed in endothelium across a wide range of tumour tissues. CLEC14A is a superior tumor endothelial marker to Robo4 in terms of specificity and level of expression on the cell surface. CLEC14A is only expressed by endothelium in the absence of shear stress and this could explain its tumor specific expression due to poor blood flow in ill-formed tumor vessels. Functional studies have shown that CLEC14A is present on filopodia and mediates endothelial cell migration and tube formation. Anti-sera to CLEC14A similarly inhibited these processes suggesting that anti-CLEC14A monoclonal antibodies would have anti-angiogenic activity. The expression profile of CLEC14A, its cell surface localisation and inhibition of pro-angiogenic activities by anti-sera make CLEC14A a uniquely interesting target.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 1589.
Abstract 1594: Targeting UBP43 for repression destabilizes PML/RARα and inhibits acute promyelocytic leukemia growth
Authors: Guo Yongli , Dolinko Andrey , Chinyengetere Fadzai , Bomberger Jennifer , Gallagher Robert , Ma Tian , Galimberti Fabrizio , Liu Xi , Sekula David , Busch Alexander , Freemantle Sarah , Dmitrovsky Ethan
Published: February 2011

All-trans-retinoic acid (RA) successfully treats acute promyelocytic leukemia (APL) at least partly through triggering degradation of the PML/RARα translocation product. Prior work revealed that the E1-like ubiquitin-activating enzyme (UBE1L) associates with the interferon-stimulated gene 15 (ISG15) to repress PML/RARα protein expression. The ubiquitin protease 43 (UBP43) removes ISG15 from conjugated proteins. We derived two different anti-human UBP43 antisera to examine UBP43 protein expression. This study explored whether RA regulates UBP43 expression and if UBP43 affects PML/RARα protein stability as well as growth or apoptosis of APL cells. Retinoid regulation of the UBE1L-ISG15-UBP43 pathway was studied in cultured NB4 APL cells, transgenic APL mice and leukemic cells harvested from APL patients. Whether the deconjugase UBP43 was a therapeutic target was determined through gain and loss of UBP43 expression experiments. This study extends prior work by reporting that RA-treatment induced UBP43 expression in RA-sensitive, but not in RA-resistant NB4 APL cells. This followed an induction of UBE1L and ISG15 expression in RA-sensitive APL cells. UBP43 functions as a negative feedback loop by reversing ISG15 conjugation of PML/RARα protein. Deregulating this loop by UBP43 knock-down repressed APL cell growth by further destabilizing the PML domain of PML/RARα protein. Notably, this triggered apoptosis in APL cells. In contrast, UBP43 over-expression stabilized PML/RARα protein and promoted APL cell growth by inhibiting apoptosis. RA-treatment of a murine transgenic APL model and of de novo cultures of APL cells from patients increased UBE1L, ISG15 and UBP43 expression. Thus, this study found that the deconjugase UBP43 was retinoid regulated in NB4 APL cells as well as in leukemic cells isolated directly from APL patients and in a mouse model for APL. Oncogenic effects of PML/RARα were reversed by UBP43 knock-down, which destabilized PML/RARα protein and triggered apoptosis in APL cells. Taken together, these findings establish UBP43 as a novel molecular pharmacologic target for APL.

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 1594.
Abstract 1598: Vasculogenic properties of triple negative breast carcinoma cells: possible implication for new molecular target
Authors: Tagliabue Elda , Plantamura Ilaria , Dugnani Erica , Ghirelli Cristina , Tortoreto Monica , Barajon Isabella , Arnaboldi Francesca , Agresti Roberto , Carcangiu Maria Luisa , Balsari Andrea , Iorio Marilena V.
Published: February 2011

Triple negative (TN) breast cancers account for about 15-20% of breast cancers and are associated with aggressiveness, poor prognosis and shorter survival.

Because of the lack of specific therapy guidelines for this subgroup there is an urgent need to define specific markers for therapeutic targeting in these tumors.

We observed that TN breast cancer cells lines are able to perform “Vasculogenic Mimicry” (VM) in vitro. In fact, all four TN cell lines evaluated (MDA-MB-231, MDA-MB-157, MDA-MB-468 and HCC1937) were able to form vascular channels when seeded on the murine tumor-derived basement membrane (Matrigel), whereas luminal (MCF7 and ZR-75-1) and HER2-positive (MDA-MB-361 and SKBr3) breast carcinoma cell lines did not exhibit vascular structures. Then, we evaluated the VM in xenograft tumors derived from MDA-MB-231, MCF7 and MDA-MB-361 using transmission electron microscopy (TEM). In MDA-MB-231 xenografts it has been revealed channel-like structures formed by tumor cells encompassing erytrocytes, whereas in MCF7 and MDA-MB-361 xenografts the endothelial lining delimiting blood vessels was clearly visible. Notably, blood vessels surrounded by tumor cells were also identified in human TN specimens processed for TEM, and these structures were significantly more frequent in TN compared to non-TN tumors. 60% reduction in TN vascular channel formation in vitro by an anti-bFGF monoclonal antibody together with no effect using anti-VEGF antibody indicated that TN breast carcinoma cells can generate vascular channels through bFGF-mediated pathway. Silencing different receptors involved in bFGF signal (i.e. FGFR2 and PDFGR) abrogated VM in TN cells.

Treatment of MDA-MB-231 xenografts with a TKI able to block both FGFR and PDGFR induced tumor regression, versus only slight growth inhibition in tumors derived from MCF7 luminal cell line.

In conclusion our findings point to the possibility that TN tumors generate vascular channels independent of tumor angiogenesis and that targeting this VM capability may represent a valid therapeutic tool. (Partially supported by AIRC).

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 1598.
Abstract 160: CpG island methylator phenotype (CIMP) and P53 methylation patterns in Puerto Rican Hispanic colorectal cancer patients
Authors: Castro Cristina I. , Lacourt Mercedes Y. , Bernabe Raul D. , Cruz-Correa Marcia R.
Published: February 2011

Introduction: The CpG island methylator phenotype (CIMP) with extensive promoter methylation seems to be a distinct epigenotype of colorectal cancer (CRC). CIMP hypermethylation blocks transcriptional activation of several genes associated with tumor suppression, cell cycle, DNA repair, differentiation and apoptosis. Moreover, the promoter CpG island hypermethylation is an important epigenetic change associated with gene silencing, poor prognosis and lower response to chemotherapy in CRC patients. However, specific clinical and pathologic features have not been associated with CIMP status in Puerto Rican Hispanic CRC patients. In this pilot study we determined the CIMP status and the methylation pattern of P53 gene in CRC to establish a possible correlation between aberrant methylation and clinicopathological characteristics.

Methods: We employed methylation-specific PCR (MSP) to evaluate DNA methylation in eight CIMP-specific promoters (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1, hMLH1, p16) and the methylation pattern of the tumor suppressor gene P53 in twenty-five CRC cases. No CIMP status was defined as no methylated locus, CIMP-Low status as one to three methylated loci, and CIMP-High status as four or more methylated loci. In addition, clinicopathological and methylation patterns were correlated to the CIMP status using Wilcoxon rank tests, Chi square and Fisher exact t-tests, as appropriate using STATA 10.0.

Results: Twenty-five CRC cases (mean age at diagnosis 64.2 ± 11.7 years; 11 males) were evaluated. Tumors were mostly located in the distal colon (76.2%), were moderately differentiated (88.8%), and were Dukes Stage B/C (86.7%). Twelve percent (3/25) of CRC cases were CIMP-High, 80% (20/25) were CIMP-Low and 16% (4/25) were No-CIMP. Aberrant methylation was not detected in the tumor suppressor gene P53. There were no significant statistical associations between CIMP status and age at diagnosis, gender, tumor staging, differentiation, tumor location or survival.

Discussion: This pilot study is the first work to evaluate the CIMP status in Puerto Rican Hispanics CRC patients. CIMP is a distinct epigenotype of colorectal cancer, and may be less frequent among Hispanic patients than previously reported in other racial and/or ethnic groups. Understanding epigenetic silencing by methylation of promoter regions of the CpG island will enable understanding of the colorectal tumorigenesis in Hispanic patients that may help tailor screening and therapeutic strategies for Hispanic patients. Current efforts are underway to characterize a large number of Puerto Rican Hispanics CRC patients.

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 160.
Abstract 1600: Molecular Characterization of PAX5 alterations in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL)
Authors: Lonetti Annalisa , Iacobucci Ilaria , Papayannidis Cristina , Ferrari Anna , Guadagnuolo Viviana , Cilloni Daniela , Messa Francesca , Paolini Stefania , Chiaretti Sabina , Soverini Simona , Saglio Giuseppe , Pane Fabrizio , Baccarani Michele , Foà Robin , Martinelli Giovanni
Published: February 2011

Background: Recently, a genome-wide analysis using single nucleotide polymorphism (SNP) microarrays, identified genetic alterations targeting PAX5 in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, the occurrence of PAX5 alterations has not been investigated in adult BCR-ABL1-positive ALL.

Aim: To characterize mutations and rearrangements on 9p involving PAX5 in adult BCR-ABL1-positive ALL.

Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing.

Results: SNP array analysis identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was heterozygous. Four patterns of PAX5 deletion were observed: focal deletion involving only the PAX5 gene in one case (3%); deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in PAX5 haploinsufficiency or altered gene expression. In one patient the deletion of PAX5 involved only a subset of exons (2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains.

To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used: genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Normal and deleted PAX5 subgroups were also investigated for point mutations, analyzing each exon by direct sequencing, but we did not found nucleotide substitutions.

Conclusion: PAX5 deletions, but not point mutations, are frequent events in adult BCR-ABL1 positive ALL and are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL.

Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus.

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 1600.
Abstract 1606: SON is a novel therapeutic target for pancreatic cancer
Authors: Furukawa Toru , Tanji Etsuko
Published: February 2011

To find novel therapeutic molecular targets for pancreatic cancer, we conducted a systematic knockdown screening of signaling-associated molecules in pancreatic cancer cells. During the course of screening, we found that knockdown of SON, a DNA binding protein of unknown function, by short interference RNA induced a strong inhibitory effect on proliferation of cultured pancreatic cancer cells in vitro. The colony formation assay employing the short hairpin RNA vector targeting SON (shRNA-SON) revealed a strong suppression of survival of cells. Flowcytometry analysis uncovered that the knockdown of SON induced G2/M arrest and apoptosis. Stable transfectants of cancer cells with shRNA-SON showed marked retardation of tumor formation in xenograft model. Knockdown of SON induced less suppressive effect on HPDE, the immortalized normal pancreatic duct epithelial cell, which indicated that the suppressive effect on proliferation was specific for cancer cells. We established 293-cell clones stably expressing EGFP-SON and found that SON was confined in speckles in intercalated nuclei and released in cytoplasm during mitosis. These results indicated that SON was a novel therapeutic target for pancreatic 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 1606.