bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2393: Isolation and characterization of circulating tumor cells in a mouse model of colorectal cancer
Authors: Das Avisnata , Whitcomb Tiffany L. , Allen Joshua E. , Dicker David T. , Peters Kristi L. , El-Deiry Wafik S.
Published: August 2012

Circulating tumor cells (CTCs) are indicative of disease burden in metastatic colorectal cancer (mCRC) and high counts are believed to adversely affect outcome. However there has been a lack of suitable mouse models to study CTCs in mCRC. Here we report the development of an orthotopic model of mCRC in athymic nude mice from which we have successfully isolated and enumerated CTCs using the FDA-approved Veridex Cellsearch technology (Table 1). So far we have injected 5 mice with a million cells each of the human CRC cell line DLD-1 within the layers of the cecal wall for tumor xenografts to grow. 200 microL of blood has been collected every 2 weeks, till the endpoint of 16 weeks or a humane endpoint, from the medial saphenous vein of the mouse for CTC detection. In our protocol, human leucocyte filters are used as a RBC source. The blood is centrifuged at 800 G for 10 minutes in a CellSave tube, the serum removed and replaced by an equal volume of PBS. Mouse blood is transferred to this tube after collection. Efforts are on to show CTCs from mice injected with tumor cells labeled with a suitable cellular dye like Cellvue. At the endpoint, after humanely euthanizing a mouse, the cecum and organs are analyzed for possible secondary spread (liver, lungs, mesenteric lymph nodes) following resection and preservation. H/E staining is performed for evidence of primary or metastatic tumor tissue. Positive tissue samples are further analysed using immunostaining for stem cell markers and prognostic markers CD133, CD26, ALDH1, Mcl-1, EFGR, Ki67. We are analyzing isolated CTCs for expression of these markers and note the extent of concordance. The mouse model should help to understand the role of CTCs in evaluating metastasis as well as to validate the use of CTCs as tools for “liquid biopsy” of tumors. Table 1. CTC counts in the mice at different time points from injection of cells. Mouse 4 is the negative control mouse of the cohort which received injection of normal saline instead of cancer cell suspension. * denotes clotted sample.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2393. doi:1538-7445.AM2012-2393
Abstract 2402: TGF beta-mediated epithelial to mesenchymal transition in non small cell lung cancer: effects on stemness, invasiveness and chemotherapy sensitivity
Authors: Pore Milind M. , Buikema Lianne , Hiltermann TJN , Kruyt FAE
Published: August 2012

The developmental program of epithelial to mesenchymal transition (EMT) can be activated in tumor cells and is implicated in the metastatic spread of tumor cells. EMT leads to loss of cell adhesion and increased motility of cells and can be detected by down regulation of epithelial makers such as E-cadherin and gain of mesenchymal ones such as vimentin and fibronectin. Interestingly, EMT has also been implicated in enhancing stemness properties of tumor cells and in causing chemotherapy resistance. The aim of this study is to examine the invasive, stem-ness and chemotherapy sensitive properties of mesenchymal and epithelial Non-small cell lung cancer (NSCLC) cells. In particular we use a TGF beta-inducible EMT model of A549 cells. A pannel of NSCLC cell lines, A549, H460, H322, SW1573, H1650 and H1299 were explored for epithelial (EpCAM. E-cadherin) and mesenhymal markers (N- cadherin, Vimentin and Fibronectin). Only A549 cells exhibit epithelial characteristics while remaning cell lines were either mesenchymal in nature or weekly expressing epithelial markers. Treatment of A549 cells with TGFbeta induced EMT as confirmed by loss of epithelial and gain of mesenchymal markers by FACS and WB analysis. This process was independent of Src since knockdown of Src did not effect EMT activation. Other kinases are currently evaluated for mediating EMT in this model. A549 mesenchymal cells (M) cells were somewhat more resistant to cisplatin and paclitaxel compared to parental A549 cells, whereas no marked difference in the response to proapoptotic TRAIL was found. Furthermore, using wound healing assays A549M cells showed higher rates of migration. Possible effects of EMT on stem cell properties in A549 cells were studied next. A549M cells displayed enhanced spheroid formation potential when cultured in serum free Neurobasal media (NBM) that was associated with an increase in Sox-2 expression. To further explore stemness of A549(M) cells, currently the expression of stem cell markers such as CD133, CD24, CD44 is examined. In conclusion, our results thus far suggest that in A549 NSCLC cells, induction of EMT by TGF beta results in chemoresistance, higher migratory potential and increased stem cell properties. The in-vitro results observed will be extended by in-vivo assays. For this purpose we are establishing orthotopic NSCLC mice models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2402. doi:1538-7445.AM2012-2402
Abstract 2405: Loss of TBK1 induces an epithelial-mesenchymal transition through upregulation of Slug in the breast cancer cell lines
Authors: Yang Kyung-Min , Kim WonJoo , Jung YunShin , Kim Seong-Jin
Published: August 2012

Estrogen receptor alpha (ER alpha) is a key regulator of proliferation and differentiation in mammary epithelium through their well-documented effects on transcription and represents a crucial prognostic indicator and therapeutic target in breast cancer. Here, we show that loss of TBK1 in ER alpha positive breast cancer cells causes an epithelial-mesenchymal transition (EMT). We found that TBK1 is downregulated in ER alpha negative breast tumors by analyzing published microarray data sets, suggesting that TBK1 might regulate ER alpha expression. To investigate this hypothesis, we made the ER alpha positive breast cancer cell lines stably expressing TBK1-specific shRNA. Knockdown of TBK1 decreased E-cadhrein expression (epithelial marker) and increased N-cadherin, vimentin, fibronectin expression (mesenchymal marker). Additionally, loss of TBK1 in ER alpha positive breast cancer cell lines leads to the downregulation of ER alpha expression. Interestingly, ER alpha repression by knockdown of TBK1 induced the upregulation of transcriptional repressor Slug, a master regulator of EMT. Because ER alpha expression is suppressed by loss of TBK1, it is suggested that TBK1 plays a role in the inverse correlation between Slug and ER alpha. Our findings indicate that loss of TBK1 expression is associated in between ER alpha expression status and invasive growth of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2405. doi:1538-7445.AM2012-2405
Abstract 2407: Snail 1 regulates invasion through uPA-uPAR and MMP-9 signaling in prostate cancer cells
Authors: Randle Diandra D. , Clarke Shineka , Odero-Marah Valerie
Published: August 2012

Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -α (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Since Snail1 can induce matrix metalloproteinases (MMP 2 and 9) which are also involved in cell invasion, we also examined MMP activity. Androgen-dependent LNCaP, and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. The levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. Gelatin zymography analysis was utilized to detect MMP-9 activity in the conditioned media. Currently, ARCaP-Snail cells are being transiently transfected with uPAR siRNA to assay for the effects of uPAR knockdown on uPA and MMP activity. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP cells. Also, MAPK inhibited MAPK expression in the cell and decreased uPA activity. In addition gelatin zymography showed higher MMP 9 activity in ARCaP-Snail overexpressing cells that was inhibited by MAPK inhibitor. Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of uPA and uPAR activities in addition to MMPs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2407. doi:1538-7445.AM2012-2407
Abstract 2467: Acidic pericellular pH increases contribution of cathepsin B to invasiveness of a human breast carcinoma cell line
Authors: Rothberg Jennifer M. , Bogyo Mathew S. , Sloane Bonnie F.
Published: August 2012

Among the non-cellular microenvironmental factors that may contribute to malignancy of solid tumors is an acidic peritumoral pH (pHe). We have shown that this induces secretion of the lysosomal cysteine protease, cathepsin B. The object of the present study was to further examine the effect of a slightly acidic pericellular pH (i.e., pHe 6.8) on degradation of collagen IV and the activity and secretion of proteolytic enzymes, in particular cathepsin B. For these studies, we used: 1) a highly malignant human breast carcinoma line, MDA-MB-231-RFP; 2) a 3D reconstituted basement membrane overlay culture model; 3) a live cell confocal microscopy assay for imaging proteolysis and detecting active cathepsin B via an activity based probe, GB123; 4) a protocol for quantification of proteolysis on a per cell basis; 5) and fluorometric real-time and plate-reader assays for measuring cathepsin B activity. The live cell proteolysis assay confirmed that degradation of collagen IV by the MDA-MB-231 cells was consistently higher at a slightly acidic pHe and was decreased by a highly selective cathepsin B inhibitor, CA074. A dramatic increase in cathepsin B secretion from MDA-MB-231 cells was observed upon exposure to an acidic pHe, as assessed by assays of conditioned media and analysis of secretion in real-time. Our results are consistent with the hypothesis by Gillies and colleagues of “acid-mediated invasion” in tumors and suggest a role for cathepsin B in promoting degradation of collagen IV and thereby tumor invasiveness in the acidic peritumoral microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2467. doi:1538-7445.AM2012-2467
Abstract 2468: Inhibition of autophagy as a potential mechanism by which pantoprazole, a proton pump inhibitor (PPI), enhances the therapeutic effect of docetaxel chemotherapy for solid tumors
Authors: Tan Qian
Published: August 2012

Purpose: Luciani and colleagues (JNCI 2004; 96:1702-13) have shown that PPIs can increase the effects of chemotherapy, most likely by modifying the acid-base balance across the cell membrane of intracellular organelles such as lysosomes. Lysosomes are required for autophagy, a process important for the survival of cells under certain forms of stress and a possible target for anticancer therapy. Here we evaluated the effect of pantoprazole (PTP) to (i) inhibit autophagy, and (ii) modify the cytotoxic effects of the anticancer drug docetaxel (DOC) in vivo and in vitro. Methods: Autophagy was evaluated by assessing both endogenous LC3B protein and with an optical LC3B fusion reporter (LC3B-GFP-RFP) that enables monitoring of autophagosome formation and fusion with the lysosome. Cytotoxicity of DOC +/−PTP was evaluated by a colony-forming assay for PC3 parental cells, and for cells transfected with shRNAs to inhibit autophagy. Growth delay of three human xenografts was evaluated following multiple doses of DOC+/−PTP. The distribution of drug effects in relation to blood vessels was quantified by γH2AX, and cleaved caspase 3. Results: PTP increased endosomal pH and inhibited autophagy in a dose and time dependent manner. PTP alone was not cytotoxic but increased the cytotoxicity of DOC for PC3 cells, with smaller effects against cells with reduced ability to undergo autophagy. Pre-treatment with PTP increased the growth delay caused by DOC in three different xengografts, with minimal increase in toxicity. Effects of DOC evaluated by γH2AX and by cleaved caspase 3 decreased with increasing distance from tumour blood vessels, but were enhanced by pre-treatment with PTP. Conclusions: PTP increases the therapeutic effectiveness of DOC in vitro and in vivo, and a potential mechanism is inhibition of autophagy. DOC is the preferred chemotherapy for men with metastatic castration-resistant prostate cancer, and we plan a phase II study of PTP + DOC in such patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2468. doi:1538-7445.AM2012-2468
Abstract 2473: Extracellular matrix density and acinus formation in breast cancer
Authors: Swamydas Muthulekha , Yang Chihchao , Stephens Amanda , Dean Delphine , Deitch Sandy , Burg Karen JL , Dreau Didier
Published: August 2012

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular the generation of polarized acinar and ductal structures. In vitro 3D cultures conditions mimic the formation and function of breast structures. Variations in the composition of the ECM have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the extracellular matrix appears also to play a role in normal mammary tissue development and in tumor formation. Here we show that the density of the ECM directly influences the number and the organization of the acini. Briefly, MCF10A cells were incubated in increasing densities of a combination Matrigel®/Collagen I matrix. Following a week incubation, the moduli distant from and close to the acinus-like formations were measured by atomic force microscopy. Additionally, the number of acinus-like structures was determined. To investigate the acinus organization, expressions of E-cadherin and laminin were estimated by immunochemistry. Results indicate that the modulus of the ECM was increased near the acinus-like structures. Also, the number of acinus-like structures decreased with the increase in Matrigel®/ collagen I density (p<0.05). Finally, based on the expression of the basement membrane protein laminin, the organization of the acinus-like structures presence was altered as the density of the ECM increased (p<0.05). These observations highlight the role of ECM density in the dynamic remodeling of breast tissues. (This works is supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2473. doi:1538-7445.AM2012-2473
Abstract 249: The role of cholesterol in ultraviolet light B-induced apoptosis
Authors: George Kimberly S. , Elyassaki Walid , Wu Qiong , Wu Shiyong
Published: August 2012

Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of programmed cell death under various stimuli. However, the relationship between cholesterol level modification and the activation of apoptotic signaling cascades upon ultraviolet B light has not been established. In this report, we demonstrate that upon UVB irradiation cholesterol levels in membrane rafts of skin cells increase, which leads to Fas aggregation in the rafts. Utilizing a continuous velocity floatation technique, we show that Fas accumulated in the lipid rafts of human melanoma M624 cells after UVB-irradiation. The subsequent events of DISC formation were also detected in the lipid raft fractions. Depletion of cholesterol by methyl-β-cyclodextrin reduces Fas aggregation, while overloading increases. Disruption of lipid rafts also prevents Daxx from dissociating from Fas in the lipid rafts, which is accompanied with a reduced apoptotic, but increased non-apoptotic death of UVB-irradiated human keratinocytes, HaCaT cells. Results indicate that cholesterol located in the plasma membrane of skin cells is required for lipid raft domain formation and activation of UVB-induced apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 249. doi:1538-7445.AM2012-249
Abstract 2491: High-dose acetaminophen with N-acetylcysteine rescue enhances the efficacy of cisplatin chemotherapy
Authors: Nguyen Tam , Neuwelt Alexander J. , Wu Jeffrey , Foreman Nicholas K. , Neuwelt Edward A. , Rapkin Louis B.
Published: August 2012

Acetaminophen (AAP) has been shown to enhance cisplatin (CDDP) cytotoxicity in hepatoblastoma cell lines. Further, one published case report demonstrated clinical efficacy of administering acetaminophen in conjunction with cisplatin and followed 6 hours later with n-acetylcysteine (NAC) rescue. In this study, atypical rhabdoid tumor (ATRT) and germinoma cells were treated with CDDP and AAP with or without NAC rescue. We then evaluated the cells with western blots for cleaved PARP and caspase 3 to demonstrate effects on apoptosis. Further, MTS assays were performed to evaluate cell viability of treated cells. To evaluate the effects of the various treatments on oxidative stress, dihydroethidium (DHE) and 2′,7′-dichlorofluorescin (DCFH) assays were performed to test for superoxide and hydrogen peroxide levels, respectively. It was demonstrated that AAP enhances CDDP cell kill and apoptosis in germinoma and ATRT cell lines. Further, both AAP and CDDP, independently and together, cause increased oxidative stress as measured by DHE and DCFE, but this oxidative stress is only reversed by NAC in the DCFE assay. This makes sense because NAC, a glutathione precursor, detoxifies peroxide but not superoxide. Further, our group has learned of five previously unpublished patients with recurrent liver tumors (hepatocellular carcinoma and hepatoblastoma) treated with AAP and CDDP with delayed NAC rescue. All patients tolerated the treatment well. Two of the five patients responded to the treatment, suggesting the potential of this approach.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2491. doi:1538-7445.AM2012-2491
Abstract 2496: Characterization of genes critical for chondro-osseous differentiation, metastasis and osteotropism in Ewing Tumors
Authors: Hauer Kristina , Burdach Stefan , Richter Günther H.
Published: August 2012

Ewing Tumors (ET) are highly malignant, mainly localized in bone tissue and are molecularly defined by ews/ets translocations. Metastatic spread in ET is frequent and hematogenous. Microarray analysis revealed BRICHOS domain containing proteins important for chondrogenic differentiation and canonical Wnt antagonist Dickkopf, Xenopus, Homolog of, (DKK) 2 critical for terminal bone development, to be over-expressed in ET. However, none of them seemed directly induced via EWS-FLI1 transcription factor activity. RNA interference of DKK2 expression in ET suppressed in vitro proliferation and decreased the ability for colony formation as well as invasive growth of ET cells, while BRICHOS proteins seemed essential for the maintenance of a precursor state of chondrogenic differentiation. Similarly, local tumor growth and metastasis of ET after DKK2 knock down was significantly decreased in Rag2-/-αC-/- mice. In an orthotopic tumor growth model DKK2 knock down in ET cells resulted in a strongly decreased capacity for invasive growth into bone tissue while RNA interference of BRICHOS proteins surprisingly increased their invasiveness when compared to controls. Microarray analysis after DKK2 knock down and subsequent validation with real-time RT-PCR revealed DKK2-mediated induction of CD44, ICAM1, Matrix Metalloproteinase (MMP) 1, and endochondral bone development important Parathyroid Hormone-like Hormone (PTHRP) in ET. Furthermore, subsequent functional assays demonstrated that invasiveness of ET cells was Matrix Metalloproteinase (MMP) 1 mediated, while knock down of one BRICHOS protein resulted in an increased MMP9 expression, indicating the critical involvement of matrix metalloproteases for the invasive ET growth behavior. Thus, DKK2 and BRICHOS domain containing proteins orchestrate and promote bone invasiveness and metastatic spread of this tumor, and provide significant conceptual progress in understanding both the pathogenesis of invasive bone growth of ET in particular and osteotropism in general.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2496. doi:1538-7445.AM2012-2496
Abstract 2497: ET-743 interferes with EWS-FLI1 driven WRN expression in Ewing sarcoma cells and sensitizes to topoisomerase I inhibitors
Authors: Segars Laura E. , Mendoza Arnulfo , Helman Lee J. , Grohar Patrick J.
Published: August 2012

The hallmark of Ewing sarcoma is the EWS-FLI1 transcription factor believed to be responsible for oncogenesis and tumor progression. ET-743 (Trabectedin, Yondelis™) is a natural product recently identified as a particularly active agent for Ewing sarcoma cells in vitro. We have recently demonstrated that ET-743 interferes with the activity of the oncogenic transcription factor EWS-FLI1. In this report, we examine the link between EWS-FLI1 suppression and the DNA damage response. We hypothesize that blocking the EWS-FLI1 associated DNA damage response may confer additional sensitivity to specific cytotoxic agents. DNA repair pathways and EWS-FLI1 targets were evaluated by microarray expression, western blot analysis and siRNA interference. Microarray expression and western blot analysis revealed that ET-743 suppresses WRN and XRCC4 expression, critical proteins in the Non-Homologous End Joining Pathway (NHEJ) repair pathway. Further siRNA data showed that the EWS-FLI1 transcription factor drives the expression of WRN. These results suggest that WRN suppression by ET-743 is occurring through an EWS-FLI1 dependent mechanism. Since the NHEJ pathway is associated with resistance to specific chemotherapeutic agents, several combination therapies were investigated in vitro. ESFT cells show heightened sensitivity to the combination of ET-743 and the topoisomerase I inhibitor, SN-38. This result was unique to topoisomerase I inhibitors and further restricted to ESFT cell lines. Consistent with these results, previous reports have established that WRN deficient cells are hypersensitive to topoisomerase I but not topoisomerase II inhibitors. Additionally, we found that irinotecan augments the ET-743 mediated transcriptional inhibition of EWS-FLI1. Taken together these mechanisms account for the heightened sensitivity of the combination of ET-743 and irinotecan observed both in vitro and in vivo in two different ESFT xenograft models. In summary, by employing a mechanism based approach, we have identified a novel combination therapy of ET-743 and irinotecan. By linking the EWS-FLI1 transcriptional program to the DNA damage response in ESFT cells, we expose an inherent weakness to topoisomerase I inhibitors thus creating a cell type specific sensitivity to the drug. This report provides a novel method of oncogenic transcription factor targeting that involves direct suppression of downstream targets and exploitation of the resulting changes in gene expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2497. doi:1538-7445.AM2012-2497
Abstract 2500: The pivotal role of p53 in doxorubicin-induced acute versus chronic cardiotoxicity
Authors: Zhu Wuqiang , Reuter Sean , Zhang Wenjun , Wagner Gregory R. , Payne Ronald M. , Caldwell Randall L. , Shou Weinian , Field Loren J.
Published: August 2012

Introduction: Doxorubicin (DOX) is an effective anticancer chemotherapeutic which also induces acute and chronic cardiotoxicities. Inhibition of p53 activity is cardioprotective against acute cardiotoxicity in adult mice receiving high doses of DOX. However, functional p53 also reduces DOX-induced mitochondrial DNA oxidation. To explain these paradoxical results, we examined the role of p53 in DOX-induced acute and chronic cardiotoxicity with a clinically relevant juvenile mouse model. Methods: Two week old MHC-CB7 mice (which express dominant-interfering p53 in cardiomyocytes, CMs) and their non-transgenic (NON-TXG) littermates were given weekly DOX injections for 5 weeks (25 mg/kg total dose). Animals were studied 1 week (acute stage) or 13 weeks (late stage) after the last DOX injection. Cardiac function was measured with echocardiography. CM apoptosis was quantified using anti-active caspase-3 staining. The level of p53, Akt (an anti-apoptotic protein), and ataxia telangiectasia mutated (ATM, a marker for DNA-damage response) was measured by western blot using whole cell and mitochondrial fractions. Results: During acute stage, left ventricular (LV) systolic function was preserved (Fractional Shortening, FS, 58 ± 2.0% vs. 45 ± 1.3%, p<0.008) and CM apoptosis was reduced (0.003 ± 0.002/mm2 vs. 0.011 ± 0.002/mm2, p<0.008) in MHC-CB7 mice vs. NON-TXG mice. DOX increased mitochondrial p53 and decreased phosphorylated Akt in NON-TXG mice, and increased phosphorylated ATM in MHC-CB7 mice. During late stage, LV systolic function in MHC-CB7 mice markedly deteriorated, reaching values similar to those in NON-TXG mice (FS = 52 ± 2.5% vs. 50 ± 2.0%, p>0.05). CM apoptosis was elevated in MHC-CB7 mice as compared to NON-TXG mice (0.076 ± 0.007 mm2 vs. 0.042 ± 0.008 mm2, p<0.008). Phosphorylated Akt and ATM were normalized during late stage. Conclusions: Although inhibition of p53 activity protects against DOX-induced CM apoptosis and preserves cardiac function during acute stage, it exacerbates CM apoptosis and cardiac dysfunction during late stage. Ongoing experiments will determine the correlation between mitochondrial DNA damage during acute stage and CM apoptosis during late stage in DOX-treated MHC-CB7 mice. Acknowledgement: This work was supported by a grant (HL85098) from National Institutes of Health (to L.J.F.). Wuqiang Zhu was supported by a postdoctoral fellowship (121101) from American Cancer Society.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2500. doi:1538-7445.AM2012-2500
Abstract 2521: Targeting CD44 in head and neck squamous cell carcinoma (HNSCC) with a new humanized antibody RO5429083
Authors: Perez Aymee , Neskey David M. , Wen Judy , Goodwin Jarrard W. , Slingerland Joyce , Pereira Lutecia , Weigand Stefan , Franzmann Elizabeth J.
Published: August 2012

Introduction: HNSCC is a debilitating and deadly disease for which current treatments are extremely toxic and ineffective. Increasing data suggest that persistent cancer stem cells (CSC), resistant to standard chemo and radiation therapy, give rise to disease recurrence. Thus, poor outcomes may reflect ineffective CSC killing. CD44 is a promising therapeutic target that has two defined roles in tumorigenesis: it is a putative cancer initiating cell or CSC marker and it promotes migration and proliferation through interaction with EGFR, Rho and Ras signaling. Therefore therapies that target CD44 may destroy the CSC population, which is resistant to therapy while precisely eradicating the proliferating population of cancer cells. Methods: RO5429083 is a novel functional monoclonal antibody, developed by Roche, that targets a glycosylated, conformation dependent epitope of CD44. To evaluate CD44 as a therapeutic target in vivo, we established CAL 27 (HNSCC cell line) xenografts in nude mice and treated them with RO5429083 (Roche) in comparison to cisplatin (DPP), a conventional drug used in HNSCC treatment. After treatment, the mice were sacrificed, and the tumors were embedded in paraffin for IHC analysis. We also analyzed the effect of RO5429083 in vitro on peripheral blood lymphocytes (PBMCs). Results: RO5429083 treatment displayed a remarkable tumor growth inhibition in CAL 27 xenografts compared to DPP or control (P<0.005). Moreover, treatment with this new humanized antibody inhibited constitutive EGFR phosphorylation on CAL 27 xenografts. In addition, treatment of PBMCs from healthy donors with RO5429083 produced an expansion of cytolytic natural killer (NK) cells. Conclusion: Based on our studies the anti-CD44 antibody RO5429083 effectively inhibits tumor growth in the CAL 27 model of HNSCC. In addition to decreasing EGFR signaling, RO5429083 appears to have a protective effect on PBMCs and expands the NK cell population. In conclusion, we anticipate that anti-CD44 therapy will effectively target the stem cell population while boosting the number of NK cells, and thus may synergize with drugs that work via antibody-dependent cell-mediated cytotoxicity (ADCC) such as Cetuximab.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2521. doi:1538-7445.AM2012-2521
Abstract 253: The cancer testis antigen MAGED2 protects human melanoma cells from TRAIL-induced apoptosis by suppressing TRAIL-R2 expression
Authors: Tseng Hsin-Yi , Chen Li Hua , Ye Yan , Jiang Chen Chen , Hersey Peter , Zhang Xu Dong
Published: August 2012

Melanoma-associated antigen D2 (MAGED2), a member of the MAGE-II cancer testis antigen family, is expressed by almost all normal tissues and many different cancer tissues. Although it has been reported that MAGE proteins have a pro-survival role in cancer cells such as malignant mast cells, the potential biological significance of MAGE proteins in melanoma remains to be established. Here, we show that MAGED2 is involved in the protection of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppressing the expression of TRAIL-R2, the major TRAIL death receptor that mediates TRAIL-induced apoptosis in melanoma. siRNA knockdown of MAGED2 sensitised melanoma cells to apoptosis induced by TRAIL. This was associated with enhanced activation of the caspase cascade and formation of the TRAIL death-inducing signalling complex (DISC), suggesting that MAGED2 acts at or above the DISC to protect melanoma cells against TRAIL-induced apoptosis. Indeed, inhibition of MAGED2 by siRNA resulted in up-regulation of TRAIL-R2 on the melanoma cell surface, which appeared to be specific, because knockdown of MAGED2 did not impinge on the expression of other TNF receptor family members, nor did it up-regulate TRAIL-R2 in melanocytes. Up-regulation of TRAIL-R2 by inhibition of MAGED2 was mediated by a p53-responsive transcriptional increase, in that p53 was also up-regulated in melanoma cells with MAGED2 knocked down, and that knockdown of MAGED2 did not up-regulate TRAIL-R2 in melanoma cells deficient in p53. In summary, our results identify MAGED2 as an important mechanism that suppresses TRAIL-R2 expression, thus protecting melanoma cells from TRAIL-induced apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 253. doi:1538-7445.AM2012-253
Abstract 2533: A novel approach to generate anti-Tn antibody for cancer diagnosis and immunotherapy
Authors: Chiang Hsiao-Ling , Lin Chi-Yu , Jan Fan-Dan , Chen Chiao-En , Wu Chia Cheng , Nieh Shin , Lin Chun-Cheng , Hwang Jaulang
Published: August 2012

Alterations in carbohydrate epitopes present on the cell surface are often hallmark of the transition from normal to neoplastic tissue. The concept of using carbohydrate antigens as cancer marker has thus spurred intense research interests into exploiting TACAs for the development of anticancer vaccines. This study presents a novel carrier protein containing a binding domain to the receptor of antigen presenting cells (APC) and cysteine-rich repeat peptide to serve as docking sites for conjugating tumor associated carbohydrate antigens (TACAs) or haptens. This novel carrier protein after conjugation with Tn, (GalNAcα1-O-Ser/Thr), can effectively elicit high titer of IgG1 antibody rather than IgM against Tn with high specificity in immunized mice. We then performed immunohistochemical (IHC) staining of twenty-six prostate cancer samples by staining with anti-Tn antibody, and we observed that the IHC staining with anti-Tn antibody is proportional to the malignancy of prostate cancer. Furthermore, we have examined the anti-Tn vaccine potency in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. TRMAP mice, which were treated with anti-Tn vaccine, showed longer survival rate and delayed tumor growth. We also successfully constructed the cDNA of mouse anti-Tn antibody from hybridoma cell line, and engineer this mouse cDNA as single-chain anti-Tn mouse antibody and anti-Tn immunotoxin. Our preliminary results showed that anti-Tn single-chain antibody can as a diagnostic antibody to distinguish normal prostatic tissue and prostate cancer, and anti-Tn immunotoxin can inhibit tumor cell growth. This study presents that our carrier protein can offer a platform for designing vaccine against tumor-associated weak immunogen and applies a new strategy in cancer immunotherapy in future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2533. doi:1538-7445.AM2012-2533
Abstract 2535: Regulation of Chk1 by the mTOR pathway is essential for cancer cells to complete DNA replication in response to replication stress
Authors: Shen Changxian , Goto Greicy H. , Oswald Duane J. , Cam Hakan , Kurmasheva Raushan , Kitagawa Katsumi , Houghton Peter J.
Published: August 2012

Oncogenic signaling leads to DNA replication stress, DNA damage and consequently DNA damage response (DDR), a barrier for tumorigenesis. For survival and proliferation, cancer cells must overcome this oncogene-induced DDR, but the molecular mechanism by which this is achieved is largely unknown. Here we report that the mTOR pathway prevents either spontaneous or drug induced DNA damage by sustaining ATR-Chk1 checkpoint in pediatric rhabdomyosarcoma (RMS) both in vivo and in vitro. Pharmacological inhibition of mTOR signaling by the selective TOR kinase inhibitor AZD8055 lead to spontaneous DNA damage and apoptosis as demonstrated by the induction of H2AX phosphorylation and PARP1 cleavage both in cell culture and tumor xenografts of RMS. Treatment RMS Rh30 cells with AZD8055 enhanced hydroxyurea induced H2AX phosphorylation and attenuated ATR-Chk1 checkpoint activation, as shown by decreased levels of phospho-Chk1(S345). We further found that mTOR signaling controls CHK1 expression at the transcriptional level via enhancing the activity of cyclin dependent kinases (CDKs). Consistent with its essential role for cells to survive DNA replication stress, siRNA-mediated knockdown of Chk1, or inhibition of Chk1 function by a Chk1 kinase inhibitor resulted in H2AX phosphorylation, while ectopic increase of Chk1 attenuated the DNA damage induced by mTOR kinase inhibition. Most importantly, the mTOR kinase inhibitor prevented the slow S-phase progression following DNA replication stress, which was accompanied with a decrease of Chk1 protein levels and phospho-Chk1(S345). Our data suggest that the TORC1/TORC2 pathway promotes RMS tumorigenesis by enhancing the cell survival functions of the ATR-Chk1 checkpoint in response to DNA replication stress. Our findings discovered an important unexpected functional linkage between TOR signaling and the ATR-Chk1 checkpoint signaling cascade and may impact targeting these two prominent cancer cell signaling pathways. Supported by CA77776.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2535. doi:1538-7445.AM2012-2535
Abstract 2594: Chamomile (Matricaria chamomilla L.) upregulates heme oxygenase-1 through activation of ERK-Nrf2 signaling: Cytoprotective mechanism against oxidative damage
Authors: Bhaskaran Natarajan , Shukla Sanjeev , Gupta Sanjay
Published: August 2012

Protection of cells from oxidative insult could be attained by directly scavenging the reactive oxygen species or more significantly by stimulating the intracellular antioxidant defense through induction of antioxidant gene expression. It has been well documented that the transcriptional activation of Nrf2 in response to oxidative stress results in increased nuclear translocation and its binding to the antioxidant response elements (AREs) found in the promoters of genes, encoding antioxidant enzymes. Studies have demonstrated that Mitogen Activated Protein Kinase (MAPK) family is important in modulating ARE driven gene expression via Nrf2 activation. In the present study, we investigated which particular MAPK family member plays an important role in the regulation of chamomile-induced Nrf2-dependent ARE activity and ARE-driven antioxidant gene expression in mouse RAW 264.7 macrophage cells. We used aqueous chamomile extract (5, 10, 20 and 40µg/mL) obtained from dried flowers with and without MAPK pharmacological inhibitors to investigate their effect on Nrf2 activation. Pretreatment of macrophages with chamomile significantly induced the expression of antioxidant gene, heme oxygenase (HO)-1 mRNA and protein in concentration and time dependent manner. Chamomile exposure increased the nuclear Nrf2 levels which correlated with Nrf2 phosphorylation and its dissociation from Keap1 in both concentration and time dependent manner. Furthermore, chamomile treatment increased the phosphorylation of ERK1/2, but not other kinases, which might result in Nrf2 phosphorylation. Treatment of macrophages with 5µM U0126 (ERK1/2 inhibitor) was able to reduce chamomile-induced ERK phosphorylation, as well as Nrf2 phosphorylation, affecting the levels of HO-1. To confirm the role of Nrf2 in the induction of HO-1, knockdown of Nrf2 using siRNA in the macrophages resulted in significant inhibition of HO-1 expression. Furthermore, chamomile protected the cells against H2O2-induced oxidative stress and this protective effect was inhibited by U0126 and ZnPP (Zinc protoporphyrin-10µM), a HO-1 inhibitor. Taken together, these data suggest that chamomile augments cellular antioxidant defense capacity through induction of HO-1 via ERK-Nrf2-ARE signaling pathway, thereby protecting the cells from oxidative stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2594. doi:1538-7445.AM2012-2594
Abstract 2595: Screening for genomic rearrangements in BRCA1 and BRCA2 genes in Algerian breast/ovarian cancer families
Authors: Cherbal Farid , Salhi Nadjet , Bakour Rabah , Adane Saida , Boualga Kada , Maillet Philippe
Published: August 2012

Background: Breast cancer is the leading cause of cancer death in women in Algeria. To date, few molecular genetics studies of BRCA1 and BRCA2 germline mutations have been reported in the Algerian population. The most frequent mutations encountered in BRCA genes are deletions or insertions of a few bases or single-base substitutions that result in premature stop codons. However, an increasing number of large genomic rearrangements (LGRs) have been identified especially in BRCA1 gene, whereas the frequency of genomic rearrangements in BRCA2 gene is low in many populations. The objective of our study was to characterize genomic rearrangements in 76 Algerian breast/ovarian cancer patients and relatives tested negative for small point mutations in BRCA1 and BRCA2 genes. Methods: MLPA was performed on genomic DNA as described by Schouten et al (2002). Screening for genomic rearrangements in BRCA1 and BRCA2 genes of 76 patients and relatives were performed by using MLPA commercial kits P002-B1, P087-B1 and PO45-B2, PO90-B2 respectively (MRC-Holland, Amsterdam, The Netherlands). To confirm the rearrangement and to determine the exact site of its genomic breakpoints, we performed long-range PCR of genomic DNA using the Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany). Results: MLPA analysis of the BRCA1 gene revealed two germline alterations in two unrelated patients among 76. A novel deletion of BRCA1 exon 2 (c.-19-α_80+−del/p.α) and a novel genomic breakpoint in deletion of BRCA1 exon 8 (c.442-α_547+−del/p≤) were identified in two patients with breast/ovarian cancer and bilateral breast cancer, respectively. No LGRs were detected in BRCA2 gene. We found by using Long range PCR technique that our patient with the BRCA1 exon 8 deletion is heterozygous for a 2.6 kb deletion. Investigations aimed at determining the genomic breakpoint of the BRCA1 exon 2 deletion described in our study is on going. Conclusion: For the first time, we report here two genomic rearrangements in BRCA1 gene in Algerian breast/ovarian cancer patients. Our results reinforce the idea that breast/ovarian cancer families tested negatively for small point mutations should be routinely screened with MLPA for large genomic rearrangements in BRCA1 and BRCA2 genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2595. doi:1538-7445.AM2012-2595
Abstract 2596: Correlation of TGF beta receptor II and pSmad2 expression with clinico-pathological factors in human breast cancer: An immunohistochemical observation from the Shanghai Breast Cancer Study
Authors: Qiu Qingchao , Su Yinghao , Lu Wei , Cai Hui , Zheng Ying , Shu Xiao Ou , Zheng Wei , Cai Qiuyin
Published: August 2012

Loss of transforming growth factor (TGF)-α expression occurs in the early stages of breast carcinogenesis, which contributes to tumor progression. The loss of TGF-β responsiveness frequently occurs at the level of the TGF- ≤ receptor II (TGF-βRII) in breast cancer. Smad2 is a major receptor-activated Smad downstream of TGF-β signaling. Phosphorylated Smad2 (pSmad2) is translocated into the nucleus to modulate transcription of target genes involved in many cell functions. We evaluated the correlation of TGF-βRII and pSmad2 protein expression with clinico-pathological factors in human breast cancer tissue from 435 breast cancer cases from the Shanghai Breast Cancer Study, a population-based, case-control study. Expression of the TGF-βRII and pSmad2 proteins was detected using a double immunofluorescence staining method, which was validated with standard single immunostains. Lab-constructed tissue microarray samples were used as positive and negative controls. The immunofluorescence signals were designated as negative, positive, or strong-positive based on a modified Allred scoring system. Staining patterns of TGF-βRII were classified into membranous or cytoplasmic predominant. TGF-βRII expression intensity was significantly higher in normal and early-stage breast cancers (in situ carcinoma) than that in later stages (in situ carcinoma adjacent to invasive carcinoma, and invasive carcinoma) (P<0.001). The cytoplasmic predominant expression pattern of TGF-βRII was more frequently observed in breast cancer than that in normal breast epithelium on the same tissue section (P<0.001). pSmad2 expression was significantly increased in breast cancer compared with adjacent normal breast epithelium. In addition, the increased pSmad2 expression was significantly associated with higher breast cancer grade (P=0.004). No significant correlations between TGF-βRII/pSmad2 protein expression and other prognostic factors, such as age at diagnosis, menopause status, history of breast cancer, TNM stage, and ER/PR/HER2 status, was found. The results of this study suggest that reduced TGF-βRII expression and its cytoplasmic predominant expression pattern may be associated with the progression of breast cancer, and increased pSmad2 expression may be associated with carcinogenesis and poor differentiation of breast cancer cells. Additional samples are being analyzed to validate these findings and to evaluate the expression of TGF-βRII and pSmad2 in relation to breast cancer survival.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2596. doi:1538-7445.AM2012-2596