bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5297: A novel role of cdc-family gene PFTK1 in the control of liver cancer cell motility
Authors: Leung Wilson K-C , Ching Arthur KK , Chan Anthony WH , Poon Terence CW , To Ka-Fai , Wong Alice ST , Wong Nathalie
Published: February 2011

Cancer metastasis remains a major cause of cancer morbidity and mortality in individuals diagnosed with hepatocellular carcinoma (HCC). We previously reported on regional chromosome 7q21-q22 gains in close association with HCC progression, and discerned a candidate proto-oncogene PFTK1 within this region by array-based CGH mapping. The PFTK1 protein, PFTAIRE protein kinase 1, is a novel member of the Cdc2-related serine/threonine protein kinases. Our earlier investigations by ectopic expression and gene knockdown of PFTK1 confirmed a functional role for the PFTAIRE protein kinase in the motile phenotype of HCC cells, yet the biological basis remained to be determined. The aims of this study are therefore to establish the clinicopathologic significance of PFTK1 in HCC, and to define the PFTK1-modulated mechanisms in the control of HCC cell motility. Recent Tissue Microarray Analysis (TMA) on 180 paired primary HCC and their adjacent non-tumoral liver suggested common up-regulated PFTK1 compared to non-malignant counterpart (76.1%; p<0.0001). Correlative analysis further indicated PFTK1 over-expression in association with advanced tumor grading (P<0.0001) and the presence of microvascular invasion (P=0.05). By 2D-PAGE coupled with mass spectrometry, comparative proteomic profiling for phosphorylated proteins in PFTK1-suppressed HCC cells highlighted 5 differentially down-regulated spots, which included β-actin (ACTB), transgelin2 (TAGLN2), heat shock protein 70, mitochondrial aldehyde dehydrogenase and 14-3-3 gamma protein. Western blot analysis further verified a consistent reduction on ACTB phosphorylation and the serine phosphorylation of the TAGLN2 protein in the absence of PFTK1 protein kinase. Immunofluorescence analysis for cytoskeletal organizations indicated marked reduction on the actin stress fibers in PFTK1 knockdown cells. In conclusion, our results suggested that PFTK1 can affect the actin cytoskeletal organization and thus a motile phenotype in HCC cells through phosphorylation of ACTB and TAGLN2

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 5297.
Abstract 5303: Systemic immunosuppression in glioblastoma: the interplay between lymphopenia, CD14+HLA-DRlo/neg monocytes, tumor factors, and dexamethasone
Authors: Gustafson Michael P. , Lin Yi , New Kent C. , Bulur Peggy A. , O'Neill Brian P. , Gastineau Dennis A. , Dietz Allan B.
Published: February 2011

Patients with glioblastoma (GBM) exhibit profound systemic immune defects that affect the success of traditional and immune-based treatments. A better understanding of the contribution of the tumor and therapy on systemic and local immune suppression is necessary to improve immune based therapies and monitor the effects of conventional and novel therapies. To identify possible sources of immunosuppression, we measured the constituents of circulating immunity in newly diagnosed and recurrent GBM patients with or without dexamethasone treatment. We identified dexamethasone dependent and independent changes in systemic immunity associated with GBM. Of note, we identified a profound lymphopenia associated with dexamethasone treatement that manifested itself in the T cell compartment with targeted effects on the CD4+ T cells. In addition, we identified tumor and dexamethasone dependent altered monocyte phenotypes. The major population of altered monocytes (CD14+HLA-DRlo/neg) inhibited T cell proliferation, was unable to fully differentiate into mature dendritic cells, was associated with dexamethasone-mediated changes in CCL2 levels, and could be re-created in vitro using tumor supernatants. Interestingly, CD14+HLA-DRlo/neg monocytes were elevated similarly in newly diagnosed and recurrent patients. These data demonstrate that both tumor and steroids contribute to systemic immunosuppression. We provide evidence that tumors express high levels of the monocyte recruitment chemokine CCL2, can contain high numbers of CD14+ cells, that tumor supernatants can transform CD14+HLA-DR+ cells into CD14+HLA-DRlo/neg immune suppressors, and that dexamethasone reduces CCL2 in vitro and is correlated with reduction of CCL2 in vivo. Consequently, we have developed a model for tumor mediated systemic immune suppression via recruitment and transformation of CD14+ 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 5303.
Abstract 5306: A20 negatively regulates immunostimulatory activity of macrophage
Authors: Wang lifeng , Hong Bangxing , Jiang Xiaoxia , Jones Lindsey , Chen Siyi , Huang Xue F
Published: February 2011

Macrophage represents a class of professional antigen-presenting cells, but usually only exhibits low level immunostimulatory activity. Here we investigate the role of A20, a negative regulator of several key proinflammatory signaling pathways, in controlling macrophage-associated immunostimulatory ability. We find that A20-silenced macrophage has enhanced expressions of costimulatory molecules, MHC complexes, and proinflammatory cytokines. As a consequence, A20-silenced macrophage displays an elevated ability to activate CD4+ and CD8+ T cell response in vitro and in vivo, and A20-silenced macrophage immunization effectively controls onset and growth of engrafted tumors. To further search the underlying mechanism whereby A20-silenced macrophage triggers potent antitumor activity, we unexpectedly found that A20-silenced macrophage induced expression of cytotoxic molecules perforin and granzyme B not only in CD8+ T cell and NK cells, but also in CD4+ T cells, and all of these cytotoxic molecule-expressing cells display a heightened cytotoxicity against tumor cells. A20-silenced macrophage induces expression of the cytotoxic molecules likely by enhancing production and signaling of IFN-g, as depletion of IFN-g by anti-IFN antibody drastically reduced expression of these cytotoxic molecules. These results indicate that A20 negatively regulates macrophage to activate antitumor immune response, and implies silencing A20 expression would lead to generation of immunostimulatory macrophage.

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 5306.
Abstract 5312: Myeloid-derived suppressor cells express the death receptor Fas and apoptose in response to T cell-expressed FasL
Authors: Sinha Pratima , Chornoguz Olesya , Clements Virginia K. , Artemenko Konstantin , Zubarev Roman A. , Ostrand-Rosenberg Suzanne
Published: February 2011

Myeloid-derived suppressor cells (MDSC) accumulate to high levels in individuals with a variety of conditions including cancer, chronic inflammation, and stress, where they are potent inhibitors of adaptive and innate immunity. Some of the factors that induce their accumulation are known; however, physiological conditions that regulate their turn-over have not been identified. Mass spectrometry analysis revealed prominent expression of apoptosis pathway proteins by MDSC and led us to hypothesize that Fas/FasL-mediated apoptosis may be a key mechanism that regulates MDSC turn-over. This hypothesis was confirmed by the findings that MDSC levels significantly regress after removal of primary mammary tumors from STAT6-deficient or CD-1-deficient mice, but do not regress in mice doubly deficient for FasL and STAT6 or CD-1. Flow cytometry and confocal microscopy studies examining activated caspase 3 expression and cytochrome C localization revealed that MDSC apoptosis is triggered via the extrinsic, and not the intrinsic, pathway. Surprisingly, activated FasL+ T cells mediated MDSC apoptosis in vivo, demonstrating a retaliatory relationship between these two cell populations, and suggesting that Fas-FasL interactions could be exploited as a strategy to reduce in vivo levels of MDSC.

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 5312.
Abstract 5314: Immunoregulatory properties of CD44+ cancer stem-like cells in squamous cell carcinoma of the head and neck
Authors: Chikamatsu Kazuaki , Takahashi Goro , Sakakura Koichi , Ferrone Soldano , Masuyama Keisuke
Published: February 2011

There is increasing evidence in certain malignancies that tumor cells are organized into a hierarchy originating from a small population of cancer stem cells (CSCs) that possesses extensive proliferative and self-renewal potential, and that these CSCs are responsible for maintaining the tumors. In squamous cell carcinoma of the head and neck (SCCHN), CD44+ cells with significant tumorigenic potential have been identified as CSCs; however, the immunological properties of such CSCs have not yet been elucidated. In this study, we investigated the immunological functions of CD44+ cells from a SCCHN cell line. Analysis of flow cytometry demonstrated that the expression of HLA-A2 molecules was deficient in the parental cell line, whereas the CD44+ cell population restored HLA-A2 expression, although at very low levels. Moreover, CD44+ cells exhibited weak HLA class II expression on the cell surface. Interestingly, down-regulation of TAP2 was found in CD44+ cells. The CD44+ cell population produced significantly higher levels of IL-8, G-CSF, and TGF-β than the CD44- cell population. Moreover, CD44+ cells have been shown to not only more strongly inhibit the proliferation of T cells activated with anti-CD3/CD28 mAb, but also to more efficiently induce CD4+CD25+FOXP3+ Treg cells and myeloid-derived suppressor cells as compared with CD44- cells. Additionally, CD44+ cells also suppressed Th1 responses (IFN-γ production by PBMCs) and enhanced regulatory T cell responses (IL-10 production by PBMCs). Thus, CSCs may have higher immunosuppressive ability promoting evasion from immunosurveillance; therefore, the development of novel immunotherapeutic strategies to efficiently overcome CSCs-driven immune suppression is urgently needed.

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 5314.
Abstract 5315: Human tumor antigen-specific helper and regulatory T cells share common epitope specificity but exhibit distinct T cell repertoire
Authors: Fourcade Julien , Sun Zhaojun , Kudela Pavol , Janjic Bratislav , Kirkwood John M. , El-Hafnawy Talal , Zarour Hassane
Published: February 2011

CD4+ regulatory T cells (Tregs) accumulate at tumor sites and play a critical role in the suppression of immune responses against tumor cells. Here, we show that two immunodominant epitopes derived from the tumor antigens (TA) NY-ESO-1 and TRAG-3 stimulate both CD4+ T-helper cells and Tregs. TA-specific Tregs inhibit the proliferation of allogenic T cells, act in a cell-to-cell contact dependent fashion and require antigen activation to suppress IL-2 secretion by T cells. TRAG-3 and NY-ESO-1-specific Tregs exhibit either a Th1-, a Th2- or a Th0-type cytokine profile and dot not produce IL-10 or TGF-β. The Foxp3 levels vary from one Treg clone to another and are significantly lower than those of CD4+CD25high Tregs. In contrast to NY-ESO-1-specific T helper cells, the NY-ESO-1-specific Treg clonotype shares a common TCR CDR3 Vβ usage with Foxp3+CD4+CD25high and CD4+CD25− T cells and was not detectable in PBLs of other NY-ESO-1+ melanoma patients and of healthy donors, suggesting that its recruitment occurs through the peripheral conversion of CD4+CD25− T cells upon chronic antigen exposure. Collectively, our findings demonstrate that the same epitopes spontaneously stimulate both T-helper cells and Tregs in patients with advanced melanoma. They also suggest that TA-specific Treg expansion may be better impaired by therapies aimed at depleting CD4+CD25high Tregs and preventing the peripheral conversion of CD4+CD25− T cells. This work was supported by NIH/NCI Grants CA90360 and CA112198 (HMZ) and Cancer Research Institute grant (HMZ).

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 5315.
Abstract 5348: Detection of herpes simplex viruses in thyroid cancer
Authors: Vasko Vasyl V. , Jensen Kirk , Patel Aneeta , Bauer Andrew
Published: February 2011

Introduction: There is limited information about the role of herpesviridae viruses in thyroid cancer.

Materials and Methods: Thyroid tissues from 109 patients with benign (44) and malignant (65) lesions were analyzed for HSV1 and HSV2 DNA. Confirmatory studies included direct sequencing, analysis of viral gene expression and activation of viral-inducible signaling pathways. Expression of viral entry receptor Nectin-1 was examined in human samples and in cancer cell lines. In vitro experiments were performed to explore the molecular mechanisms underlying thyroid cancer cell susceptibility to HSV.

Results: HSV was detected in 43/109 (39.4%) of examined samples. HSV positive tumors were characterized by interferon-beta expression, nuclear NFkB expression and Akt activation. Lymphocytes infiltration and oncocytic cellular features were common in HSV positive tumors. HSV1 was detected with same frequency in benign and malignant thyroid tumors. HSV2 was significantly associated with papillary thyroid cancer and presence of lymph nodes metastases. Nectin-1 expression was increased in thyroid tumors compared to normal thyroid tissue and further increased in papillary thyroid cancer. The level of Nectin-1 expression in cancer cell lines correlated with their susceptibility to HSV. Inhibition of PI3K/AKT or MAPK/ERK signaling did not affect the level of Nectin-1 expression but decreased thyroid cancer cell susceptibility to HSV. Thyroid cancer cell with epithelial-like morphology underwent epithelial to mesenchymal transition after chronic exposure to TNF. Induction of epithelial to mesenchymal transition was associated with increased thyroid cancer cell susceptibility to HSV.

Conclusion. HSV was detected in human thyroid tumors and HSV2 was associated with metastatic thyroid cancers. In vivo and vitro data showed that during tumor progression thyroid cells acquire increased susceptibility to HSV due to 1) increased expression of viral entry mediator Nectin-1; 2) activation of mitogenic signaling and 3) induction of EMT in metastatic cancers.

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 5348.
Abstract 5352: A high percentage of Merkel cell carcinomas is biologically associated with Merkel cell polyomavirus
Authors: Speel Ernst-Jan M. , Schmidt Andreia , Baumann M , Haesevoets Annick , Diebold J , Hofmann P , Kurrer Michael , Willi N , Offner F , Sauter Guido , Zsikla V , Cathomas Gieri
Published: February 2011

Merkel cell polyomavirus (MCPyV) DNA has been detected by PCR in 75-100% of Merkel cell carcinomas (MCC), an aggressive neuroendocrine skin cancer. MCPyV is a 5.4 kb DNA virus that expresses tumor (T) antigen in tumor tissues. The aim of this study was to analyze which subset of MCC is biologically associated with MCPyV using various viral detection methods.

Genomic DNA isolated from paraffin-embedded tissue of 61 MCC was analyzed by MCPyV-specific PCR. Using a 5kb MCPyV DNA probe, the physical status of MCPyV was determined by fluorescence in situ hybridization (FISH) using tyramide signal amplification and ApoTome microscopy. Expression of large T antigen was assessed by immunohistochemistry (IHC) using monoclonal antibody CM2B4 (kindly provided by Y. Chang).

A total of 44 of 61 (72%) MCC were positive for MCPyV by PCR. 34 of these 44 cases (77%) were also positive for MCPyV by FISH and 28 (64%) showed strong nuclear large T antigen expression by IHC. In contrast, none of the 17 PCR-negative tumors showed FISH signals and only 1 case showed a weak immunostaining for large T antigen. FISH analysis showed in 16 cases (47%) tumor nuclei with a single punctate signal indicating viral integration. In 8 of these tumors also areas were observed with a granular pattern with >1 nuclear signals varying significantly in size and intensity, indicating the presence of episomal viral DNA and/or RNA. In the remaining 18 cases (53%) an exclusive granular FISH pattern was observed.

Our data indicate that a subset of MCC is caused by MCPyV as determined by PCR, FISH and IHC analysis (46% is positive with all 3 detection methods). FISH and IHC showed a strong positive and negative concordance. FISH signal evaluation furthermore suggests viral integration in half of the positive MCC and a non-clonal viral persistence in the remaining positive cases. Further studies have to validate these findings and elucidate the molecular mechanisms underlying MCV-positive and -negative MCC development.

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 5352.
Abstract 5361: Progesterone alters cell survival and cell death pathways in estrogen-induced mammary carcinogenesis
Authors: Arumugam Arunkumar , Lakshmanaswamy Rajkumar
Published: February 2011

Breast cancer is one of the most frequent and common malignancy in women. The incidence of breast cancer is high in postmenopausal women and majority of these cancers are estrogen receptor positive. Epidemiological data suggests that estrogen plus progesterone hormone replacement therapy increases the risk of breast cancer in postmenopausal women. Further, estrogen alone therapy did not increase the risk instead decreased the risk of breast cancer in postmenopausal women. Till date the role of progesterone in breast cancer is not well understood. Our earlier studies using ACI rats have clearly demonstrated the significant role progesterone plays in the estrogen-induced mammary carcinogenesis. We now, aim to investigate the mechanisms by which progesterone promotes estrogen-induced mammary carcinogenesis in ACI rats. We chose to use the ACI rats as it is a good model to study hormone-induced mammary carcinogenesis. In brief, seven weeks old female ACI rats were divided into the following groups: 1) untreated control, 2) rats treated with 30 mg estradiol, 3) rats treated with 30 mg progesterone, 4) rats treated with 30 mg estradiol plus 30 mg of mifepristone (a progesterone inhibitor). All hormone treatments were given for 30 days by subcutaneous implants of silastic capsules. At the end of the treatment, mammary glands were excised and used for molecular analysis. Cell survival, cell death pathways, and hormone receptor status were analyzed using different molecular techniques. Expression patterns of mRNA and proteins involved in major cell survival and apoptotic pathways were significantly altered in response to progesterone inhibition. Our data demonstrates that progesterone plays a prominent role in regulating key regulatory pathways in estrogen-induced mammary carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5361.
Abstract 5362: Mechanisms involved in hormone promoted growth of preneoplastic mammary lesions
Authors: Arumugam Arunkumar , Lakshmanaswamy Rajkumar
Published: February 2011

Carcinogenesis is a complex process, which involves initiation, promotion and progression. Copenhagen strain of rat is a well known animal model which is resistant to carcinogen-induced mammary carcinogenesis. In our laboratory, we were able to induce a very high incidence of mammary cancers in these rats using combination of carcinogen N-methyl-N-nitrosourea (MNU), followed by estradiol and progesterone treatment. We have demonstrated that the resistance to mammary carcinogenesis in these rats is not due to inhibition of initiation but due to reduced hormone promotional environment and we further extend our research to investigate the molecular mechanisms involved behind this hormone-induced promotion. We used both in vivo and in vitro experimental system to understand the mechanisms involved in mammary cancer promotion and progression. Seven week old female Copenhagen rats were administered a single dose of MNU at a dose of 50 mg/kg body weight intraperitoneally. Four weeks post carcinogen administration, the rats were killed and preneoplastic lesions were excised and used for further in vitro experiments. The preneoplastic lesions were cultured and were treated with estradiol, progesterone or a combination of both hormones. Changes in survival and cell death pathways in the cultured preneoplastic lesions in response to different hormone treatments were analyzed using different molecular techniques. Our result indicates that hormone treatment induces alterations in cell survival and cell death pathways of the preneoplastic lesions demonstrating the significance of hormones in promotion and progression of 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 5362.
Abstract 5365: Zeranol-containing serum (Z-Sera) harvested from 30-day Z-implanted heifers induce transformation of human normal breast epithelial cells through down-regulation of the protein tyrosine phosphatase γ (PTPγ) tumor suppressor gene
Authors: Lin Young C. , Ye Weiping , Xu Ping-Ping , Threlfall Walter R. , Li Hong , Lin Shu-Hong , Kuo Chieh-Ti
Published: February 2011

The morbidity of breast cancer ranks first in the U.S. it remains the second leading cause of maligancy-related death in women, regardless of advances in novel therapeutic strategies. Many risk factors have been associated with breast cancer initiation, promotion and progression. Recently, Zeranol (Z), one of six growth promoters approved by FDA for use in the beef industry may cause health concerns due to the consumption of beef products containing bio-active Z metabolites. Our laboratory previously demonstrated that Z down-regulates the expression of estrogen-regulated PTPγ in primary cultured human normal breast epithelial cells and transforms the normal human breast epithelial cell line, MCF-10A, to neoplastic breast cancer cells. In the current study, we investigated the biologically active Z metabolites present in Z-Sera harvested from 30-day post Z implanted heifers (72 mg pellet) on MCF-7 cell growth and PTPγ mRNA expression in MCF-10A cells using non radioactive cell proliferation and real time PCR. Our results showed that Z-Sera at 0.5, 2.5 and 12.5% significantly increased the growth of MCF-7 cells by 42, 71, and 108%, respectively. While the sera harvested from the control heifers did not increase MCF-7 cell growth. Treatment with 2.5% Z-sera harvested 30, 60, 90 days post Z-implantation significantly down-regulated the expression of PTP γ mRNA in MCF-7 cells to 18.2, 36.5 and 51%, respectively. Furthermore, neoplastic transformation of MCF-10A cells resulted after exposure of 2.5% Z-Sera in culture medium to MCF-10A for 21-day. The expression of PTPγ mRNA, in transformed MCF-10A cells, was significantly reduced by 83, 96 and 97%, as compared to the controls. We hypothesize that the transformation of MCF-10A cells by bio-active Z metabolites contained in 2.5% Z-sera may be mediated through the down-regulation of estrogen-regulated PTPγ in MCF-10A cells. We have reported that the growth rate of pre-adipocytes isolated from the beef cattle 30-day post Z-implantation was 12 fold faster than the pre-adipocytes isolated from of the control beef cattle. In summary, our in vitro data suggest that potential adverse health risk may result from consuming beef products containing bio-active Z metabolites. (Supported by NIH grant R01 ES 015212).

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 5365.
Abstract 5378: Targeting CXCL12/CXCR4 signaling axis for pancreatic cancer therapy
Authors: Singh Seema , Talwar Sudha , Srivastava Sanjeev , Braune Sarah , Owen Laurie B. , Singh Ajay P.
Published: February 2011

Pancreatic cancer is a lethal malignancy with an extremely poor prognosis. Furthermore, it is also highly resistant to currently available therapeutic modalities. Hence, it is very important to better understand the drug-resistance mechanisms and to develop novel and effective therapeutic strategies. Chemokine signaling has been shown to play an important role in cancer progression and metastasis. Importantly, a chemokine receptor, CXCR4, has been shown to be expressed in pancreatic cancer tissues, precancerous lesions, and cancer stem cells. CXCR4 signaling induced by binding to its ligand (CXCL12) potentiates pancreatic tumor cell growth, migration and invasion, and distant metastasis in an autocrine and/or paracrine fashion. CXCL12/CXCR4 signaling has also been shown to facilitate the tumor-microenvironment interaction and induce desmoplastic alterations in the stroma, which is a common feature of pancreatic adenocarcinoma. Therefore, targeting CXCR4, alone or in combination with cytotoxic drug, might be an effective strategy for pancreatic cancer therapy. Here, we examined the expression of CXCR4 in a panel of twelve pancreatic cancer cell lines and pancreatic cancer tissues. Our data document that CXCR4 is expressed by all pancreatic cancer cell lines and overexpressed in pancreatic cancer tissues as compared to corresponding normal pancreas. Furthermore, treatment with CXCL12 induced the growth of pancreatic cancer cells, indicating the functionality of the CXCL12/CXCR4 signaling axis and suggesting its potential suitability as a therapeutic target. Interestingly, pancreatic cancer cells treated with CXCL12 showed reduced cytotoxicity to the cytotoxic drug 5-fluoro-uracil (5-FU) as compared to cells treated with drug alone. Treatment with CXCR4 antagonist, AMD3100, arrested the CXCL12-induced pancreatic cancer cell growth, and resulted in a cooperative growth inhibitory effect with 5-FU treatment. Taken together, our findings suggest that targeting of CXCL12/CXCR4 signaling axis can be exploited as a novel and effective approach for pancreatic cancer therapy, alone or in combination with cytotoxic chemotherapeutic agents.

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 5378.
Abstract 5379: Mechanism of fludarabine and oxaliplatin combined activity depends on the activity of XPF endonuclease
Authors: Zecevic Alma , Ewald Brett , Sampath Deepa , Wierda William , Plunkett William
Published: February 2011

We set out to determine the mechanism by which fludarabine and oxaliplatin exert more than additive cell killing in chronic lymphocytic leukemia (CLL). We postulate that enhanced activity of oxaliplatin and fludarabine combination is based on termination by fludarabine of DNA re-synthesis associated with nucleotide excision repair (NER) following removal of oxaliplatin adducts. The blocked repair would result in single-strand and double-strand DNA breaks which would activate cell DNA damage signaling and apoptosis. More than additive apoptotic killing was observed in 49% (±22) of CLL cells after exposure to oxaliplatin and fludarabine in vitro, although single agents showed minimal increase in cell death (3% ±9 for oxaliplatin, 13% ±14 for fludarabine), as demonstrated by Annexin V and PI staining at 24 hr (expected vs. observed p=0.0003; n=10). Oxaliplatin induced a 2-fold increase in DNA synthesis in CLL cells, which was inhibited by fludarabine, as determined by thymidine incorporation (n=5). Knockdown of XPF, the NER 5′ endonuclease, in CLL patient samples (n=3) eliminated oxaliplatin induced DNA synthesis (3-fold increase in thymidine incorporation with scramble siRNA compared to 1.4-fold increase with XPF siRNA). Wild type CHO (AA8) cells showed more than additive killing after combination treatment similar to the observation with CLL patient samples (predicted survival 50%; observed 21%). In contrast, only additive killing was observed in CHO cells lacking XPF (UV41) with equitoxic doses of oxaliplatin and fludarabine (predicted survival 56%; observed 54%) suggesting that XPF function is essential for the cooperative interaction of the combination. Alkaline assays demonstrated that inhibition of repair by fludarabine was accompanied by DNA strand break formation within 12 hr (n=4) in CLL patients samples. The increase in Olive tail moment ranged from 2- to 5-fold, confirming induction of single-strand breaks. The double-strand break repair proteins ATM, SMC1, H2AX and DNA-PKcs were phosphorylated as early as 2 hr post-treatment with the combination and persisted up to 24 hr (3-fold, 24-fold, 10-fold and 10-fold increase, respectively; n=4). This response suggests that DNA damage generated by fludarabine and oxaliplatin exposure was processed to double-strand DNA breaks in CLL cells. Our work provides evidence that the combination of oxaliplatin and fludarabine results in the formation of toxic DNA damage and that their combined activity depends on a functional NER pathway.

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 5379.
Abstract 538: Mesenchymal stem cell subset prevents cycling of KG1a leukemic cells by cell-cell interaction
Authors: Donepudi, SreeKanth , Porada Christopher D. , Zanjani Esmail , Almeida-Porada Graça
Published: February 2011

The interactions that occur between leukemic cells and the microenvironment are currently poorly understood. The definition of novel pathways that deliver survival and drug resistance signaling from the milieu to leukemic cells may lead to the discovery of new drugs that are able to target microenvironmental cues perpetuating the leukemic clones. Since the bone marrow (BM) environment is comprised of a complex assortment of different cell types, matrices and soluble and membrane-bound factors, it is crucial to start delineating specific cell-cell interactions and their effects on leukemic cell survival. Mesenchymal stem cells (MSC) are a fundamental component of the BM niche as they support hematopoiesis and are able to differentiate into neighboring cellular types. Therefore, in these studies we investigated the effect of Stro-1+ MSC on the growth and survival of KG1a, a primitive, differentiation-resistant subtype of acute myeloid leukemia. MSC and KG1a cells were co-cultured for 3 days under contact or non-contact conditions, at incremental ratios of 1:1, 1:5, 1:10 and 1:100 respectively, while maintaining a constant initial total number of cells for each dilution (n=3). In transwell non-contact cultures, KG1a cells proliferated at the same rate as KG1a alone with a 3 to 5 fold increase in cell numbers, regardless of the ratio. By contrast, when KG1a were cultured in direct contact with MSC, KG1a growth inhibition was directly proportional to the MSC:KG1a ratio. Specifically, MSC cultured with KG1a cells at ratios of 1:1, 1:5, 1:10, and 1:100 inhibited the growth of KG1a cells by 84%, 76%, 74%, and 45%, respectively while remaining 99-100% viable. Thus, we next performed cell cycle analysis using flow cytometry and found that while the cell cycle distribution of KG1a cultures were: 23% in G0/G1; 19% in G2/M; and 58% in S phase, KG1a co-cultured with MSC had undergone cell cycle arrest in S-phase (98%). Although our results contrast with prior studies showing that MSC inhibited KG1a in G1phase through the production of soluble factors, it is possible that the differences seen are due to the population of MSC used. Within the MSC, Stro-1+ cells constitute a primitive population of cells in intimate relation with the osteoblastic niche, the role of which is to retain stem cells in a quiescent state. Therefore, studies are currently underway to identify molecules responsible cell cycle arrest upon KG1a/MSC interaction.

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 538.
Abstract 5383: Cucurbitacin B, a novel in vivo potentiator of gemcitabine with low toxicity in the treatment of pancreatic cancer
Authors: Iwanski Gabriela B. , Thoennissen Nils H. , Lee Dhong Hyung Tony , Doan Ngan B. , Castor Brandon , Le Alison , Toh Melvin , Bokemeyer Carsten , Said Jonathan W. , Koeffler H. Phillip
Published: February 2011

Pancreatic cancer is a highly aggressive malignancy, and improvement in systemic therapy is necessary to treat this frequently encountered metastatic disease. The current targeted agents used in combination with gemcitabine improved objective response rates, but with little or no improvements in survival and also increased toxicities in pancreatic cancer patients. Recently, we showed that the triterpenoid cucurbitacin B inhibited tumor growth in pancreatic cancer cells by inhibition of the JAK/STAT pathway, and synergistically increased antiproliferative effects of gemcitabine in vitro. The anti-tumor and toxicity effects of cucurbitacin B in combination with gemcitabine were tested against human pancreatic cancer cells in a murine xenograft model. The combinatory therapy of cucurbitacin B and gemcitabine at relatively low doses (0.5 mg/kg and 25 mg/kg, respectively) resulted in highly significant tumor growth inhibition of pancreatic cancer xenografts by up to 79% (p<0.001). Remarkably, this therapy was well tolerated by the animals, including histology of inner organs, analysis of serum chemistries, full blood counts, and bone marrow colony numbers. Western blot analysis of the tumor samples of mice who received both agents, cucurbitacin B and gemcitabine, revealed stronger inhibition of Bcl-XL, Bcl-2 and c-myc, and higher activation of the caspase-cascades compared to single agent treated mice. The in vivo combination therapy of cucurbitacin B and gemcitabine has profound antiproliferative effects against human pancreatic cancer cells, without any significant signs of toxicity. This promising combination should be examined in therapeutic trials 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 5383.
Abstract 5391: Rosiglitazone and Gemcitabine combination therapy limits tumor progression and prolongs survival in pancreatic adenocarcinoma
Authors: Bunt Stephanie K. , Hollingsworth Michael A.
Published: February 2011

Pancreatic ductal adenocarcinoma (PDA) is a leading cause of cancer related mortality with a dismal 2-5% 5-year survival rate because clinically apparent disease is usually detected late in disease progression and lacks effective therapies. The current ‘standard of care’ for pancreatic cancer patients is Gemcitabine, a deoxycytidine analog, which has been shown to increase quality of life and the median survival of patients by a few weeks. Although Gemcitabine therapy limits pancreatic tumor progression, PDA is often insensitive to chemotherapeutic agents, limiting their success and underlining the need for additional therapies which would enhance the efficacy of current treatments.

We investigated combinations of Gemcitabine with drugs that target both the growing tumor and the immunosuppressive inflammatory microenvironment. Inflammation accompanies tumor progression in pancreatic cancer which can support tumor cell survival and growth directly, enhance tumor cell resistance to Gemcitabine and limit anti-tumor immune responses through induction of immunosuppressive mechanisms. Rosiglitazone Maleate, an FDA-approved drug for the treatment of type II diabetes, targets the PPARγ receptor, a major modulator of the inflammatory response. Rosiglitazone has been shown to limit tumor growth through effects on angiogenesis, differentiation and apoptosis. Additionally, some evidence suggests that PPARγ may regulate multi-drug resistance and may therefore limit tumor chemoresistance.

We found that Rosiglitazone enhanced the efficacy of Gemcitabine by reducing tumor proliferation, limiting invasiveness, and enhancing tumor cell apoptosis in vitro. Therapeutic combination of Rosiglitazone and Gemcitabine was evaluated in subcutaneous and orthotopic models of pancreatic cancer, and revealed that the combination significantly reduced tumor progression and metastasis, enhanced tumor cell apoptosis in vivo and significantly extended overall survival compared to Gemcitabine alone. Together these results provide pre-clinical data in support of combining Rosiglitazone and Gemcitabine as a clinical therapy for pancreatic adenocarcinoma.

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 5391.
Abstract 5393: Regression of a human T-cell leukemia lacking the methylthioadenosine phosphorylase (MTAP) gene without toxicity of 6-thioguanine (6TG) by pretreatment with methylthioadenosine (MTA)
Authors: Bertino Joseph R. , Johnson-Farley Nadine , Perez Raymond P. , Lubin Adam , Lubin Martin
Published: February 2011

The MTAP gene, at chromosomal locus 9p21, is frequently co-deleted with p16, especially in T-cell acute lymphocytic leukemias, mesotheliomas, gliomas, lung and pancreatic carcinomas, and sarcomas. In normal cells, MTAP cleaves MTA to adenine and 5-methylthioribose-1-phosphate, which are converted to adenine nucleotides and methionine. We previously showed that mice are protected from 6TG toxicity by preadministration of MTA, generating adenine and blocking conversion of 6TG to its nucleotide. (Lubin M, Lubin A. 2009: Proceedings of the American Association for Cancer Research. Abstract LB-29; Selective Killing of Tumors Deficient in Methylthioadenosine Phosphorylase: A Novel Strategy: PLoS One, ePub May 29, 2009). We now demonstrate that MTA completely protects mice bearing MTAP-negative CEM-CCRF human T-cell leukemia from 6TG toxicity while the tumor remains responsive. Following two small pilot studies, 24 NOD-SCID mice were inoculated subcutaneously with 5 million CEM cells; when tumor size reached 500 cubic mm, mice were randomized to 4 groups of 6 animals. All mice treated with vehicle or MTA alone were sacrificed on day 14 because of tumor size; MTA showed no toxicity. Animals treated with 6TG (75 mg/kg), on days 1, 4, 7, had marked tumor regression but died of toxicity by day 12. However, animals treated with MTA (100 mg/kg) followed one hour later by 6TG (75 mg/kg) had complete tumor regression by day 14, and no weight loss; but tumor recurred in all animals by day 24. While further optimization of dose and schedule is needed, these studies clearly show that marked tumor regression can be obtained without toxicity in tumors lacking MTAP, by competitively inhibiting 6TG conversion to nucleotides. As MTA has been given safely to humans, we are planning clinical trials using this strategy in patients with tumors lacking MTAP.

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 5393.
Abstract 5398: Drug combination synergy quantification in animals and clinics using computerized combination index method
Authors: Chou Ting-Chao
Published: February 2011

Drug combination has been used for thousand of years, and currently widely used in the treatments of cancer, AIDS, and other diseases. Drug combination studies have been carried out mostly in vitro but rarely in animals and clinics due to cost, efficiency, ethical considerations, etc. However, the “definition of synergy” is universal and can be quantitatively determined as indicated by Chou & Talalay in (i). Adv. Enz. Regul. 22: 27-55, 1984 and by Chou in (ii). Pharmacol. Rev. 58: 621-681, 2006, (iii). Leukemia & Lymphoma 49: 2059-2080, 2008, and (iv). Cancer Res. Perspective Article 2009 (in press). By definition, synergism is more than the additive effect, however, additive effect cannot be determined by the arithmetic sum of effect, nor by the fractional product method, nor by statistics, p value, but rather by the combination index (CI) where CI = 1, < 1, and > 1 indicate additive effect, synergism, and antagonism, respectively. The theory, experimental design, and computerized simulation have been discussed or illustrated in (i-iii), and in (iv) outlined the pitfalls, common errors, and frequently asked questions. The CI method of Chou-Talalay has been cited in over 400 journals and in (i) alone has been cited over 1,916 times with citations over 1,000-fold higher than the journal's impact number. The CI method, based on the median-effect principle of the mass-action law, is quantitative, efficient, simple and economical which allows using small number of data points. This paper will illustrate how synergism of two anticancer drugs can be determined in nude mice bearing mammary carcinoma MX-1, and why two similar anti-HIV clinical trials, where both used surrogate markers and fractionated doses, were published and had different results; one used 366 patients (N. Engl. J. Med. 333: 1662-1669, 1995) but was unable to determine synergism due to the experimental design, and another, which applied the CI method, used only 36 patients (Antivir. Ther. 1: 77-88, 1996), and yet was able to quantitatively determine synergism.

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 5398.
Abstract 54: Role of aldehyde dehydrogenase 1 in breast cancer stem cell survival
Authors: Zhang Min , Hogan Dale , Reddy Aramati Bindu , Ramana Kota , Srivastava Satish , Ansari Naseem H.
Published: February 2011

Tumor initiation and progression are mainly driven by a small subset of stem cells- like cancer cells (CSCs), which represent <1 % of malignant breast tumors. Most importantly breast CSCs are highly resistant to radiation- and chemo-therapy. Such resistance could be attributed to the high expression of the enzyme, Aldehyde dehydrogenase I (ALDH1), which in recent years has been suggested to be a prognostic marker of breast cancer. However, the mechanism(s) of ALDH1's involvement in CSCs’ function and resistance to radiation/chemotherapy (oxidative stress) and its role in breast cancer progression are not known. Toxicity of most of the chemotherapeutic drugs is known to be via increased oxidative stress which generates lipid aldehydes such as 4-hydroxynonenal (HNE), a potent apoptotic agent. In CSCs the levels of ALDH1 are much higher than the normal breast cell as well as cancer cells. It is likely that the resistance of CSCs to chemotherapeutic drugs is due to increased levels of ALDH1 which is known to detoxify HNE generated by oxidative stress caused by chemotherapeutic drugs. Increased HNE could kill the cells by causing increased apoptosis. To test this hypothesis we inhibited ALDH1 in the tumoregenic human breast cancer cells, MDA-MB-468, by diethylaminobenzaldehyde (DEAB) and measured HNE-induced toxicity by evaluating the levels of protein-HNE adducts which, as previously demonstrated by us, increase in oxidation-induced apoptosis. ALDH1hi/CD44+ and ALDH1low/ CD44+ cells were sorted by flow cytometry using ALDEFLUO-FITC & mouse anti-Human CD44-APC. The DEAB-treated cells when subjected to oxidative stress by H2O2, showed increased formation of protein -HNE adducts with a concomitant increase in apoptosis and decrease in cell viability. Furthermore, our results also displayed increased activation of the transcription factor, NF-kB which could be responsible for up-regulating the death genes leading to apoptosis. In addition, four specimens of human malignant breast cancer tissues and two of benign breast tumors were analyzed by double immunostaining. Compared to benign tumors, elevated levels of ALDH1 and CSC-like cells (high ALDH1/high CD44/lowCD24) were observed in the breast cancer tissues. Furthermore, levels of ALDH1 in these specimens appeared to be proportional to the grade of cancer. Our results thus suggest that disrupting ALDH1-mediated HNE detoxification in CSCs could be a novel preventive approach against breast cancer progression and metastasis.

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 54.