bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4740: Doxorubicin eliminates tumor-induced myeloid-derived suppressor cells and enhances T-helper lymphocyte-based immunotherapy in a murine breast cancer model.
Authors: Alizadeh Darya , Trad Malika , Katsanis Emmanuel , Larmonier Nicolas
Published: August 2014

Myeloid-derived suppressor cells (MDSC) represent a heterogeneous population of cells equipped with the ability to inhibit T lymphocyte-mediated immune responses. A significant increase in the number of MDSC has been reported in the blood, secondary lymphoid organs and tumor beds in tumor-bearing animals and in patients with many types of cancers. MDSC frequency correlates with the disease stage and prognosis. These cells impair CD8+ cytotoxic T lymphocyte (CTL)-mediated anti-tumor immunity by different overlapping mechanisms such as reactive oxygen species or immunosuppressive cytokine production. Importantly, MDSC elimination or inactivation substantially enhances the efficiency of immunotherapy. Multiple studies have lent support to the concept that some anti-neoplastic molecules, besides their conventional direct cytotoxic influence on cancer cells, may also exhibit immunostimulatory effects, thereby promoting anti-tumor immunity. We present results demonstrating that doxorubicin, utilized in multiple chemotherapeutic regimens and known to trigger an immunogenic type of cancer cell death, dramatically reduces the number of tumor-induced MDSC in the murine mammary tumor model 4T1. Elimination of these suppressive cells occurs by an apoptotic process. In addition, residual MDSC isolated from doxorubicin-treated animals are significantly impaired in their immunosuppressive function. Of particular interest, the number of apoptotic CD4+ or CD8+ T lymphocytes is not augmented in mice administered with doxorubicin compared to untreated animals. We further demonstrate that doxorubicin-mediated elimination of MDSC is associated with enhanced T and NK cell activation, proliferation and expression of Perforin and Granzyme B. Lastly, we evaluated a chemoimmunotherapeutic strategy associating doxorubicin with T helper-1 or T helper-17 lymphocytes generated and activated in vitro. Our results indicate that this combination regimen hinders tumor development as evidenced by reduced number of 4T1 metastatic nodules in the lungs of treated animals. This therapeutic effect is associated with an increased cytotoxic activity of CD8+ T lymphocytes in the spleen of the treated mice. These observations thus highlight a new application of doxorubicin as a MDSC depleting agent and further advocate for the implementation of this drug in cancer immunotherapy approaches.

Citation Format: Darya Alizadeh, Malika Trad, Emmanuel Katsanis, Nicolas Larmonier. Doxorubicin eliminates tumor-induced myeloid-derived suppressor cells and enhances T-helper lymphocyte-based immunotherapy in a murine breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4740. doi:10.1158/1538-7445.AM2013-4740
Abstract 4741: Inhibition of TNBC cell growth by CSPG4-specific mAb 225.28 with a Sonic Hedgehog pathway inhibitor.
Authors: Wang Yangyang , Sabbatino Francesco , Ferrone Soldano , Wang Xinhui
Published: August 2014

In the US, about 10-20% of breast cancers are found to be triple-negative breast cancer(TNBC). Because of the lack of estrogen receptor (ER) and progesterone receptor (PR) expression, and human epidermal growth factor receptor 2 (HER2) amplification, TNBC does not benefit from the currently available targeted therapies. Furthermore, recent molecular analysis of TNBC cell lines and lesions has suggested that these tumors might be enriched with cancer initiating cells (CICs). This possibility is consistent with the failure of conventional therapies to control disease progression, because according to the cancer stem cell theory, CICs play a major role in disease recurrence and metastatic spread, the two major causes of patient mortality. These findings and the lack of curative therapy for any TNBC patient with metastatic disease emphasize the need to develop effective novel therapies for this type of breast cancer. To address this need, we have developed a combinatorial immunotherapy, which eliminates both differentiated TNBC cells and TNBC CICs. The tumor antigen we have selected as a target is chondroitin sulfate proteoglycan (CSPG)4, since this membrane bound tumor antigen is expressed at high levels on both differentiated TNBC cells and TNBC CICs. CSPG4 plays a crucial role in the biology of TNBC cells, since it is involved in ERK and FAK signaling pathways which are associated with TNBC cell proliferation and migration. Furthermore, it is upregulated on activated pericytes in the tumor microenvironment. CSPG4-specific mAb 225.28 inhibits TNBC cell growth and migration. Moreover, it inhibits signaling pathways associated with TNBC cell growth and survival. It also can selectively inhibit neoangiogenesis in the tumor microenvironment, contributing to the elimination of TNBC cells, even those that do not express CSPG4. To enhance the targeting of TNBC CICs, immunotherapy is combined with the administration of an inhibitor (cyclopamine/ LDE225) of the Sonic Hedgehog Homolog (SHH) pathway which is activated in TNBC cells and, especially in TNBC CICs. Inhibition of TNBC CIC growth by mAb 225.28 is around 10%, but is enhanced up to about 70% when mAb 225.28 is combined with a SHH pathway inhibitor. Our in vitro findings strongly suggest that targeting CSPG4 with mAb 225.28 in combination with a SHH inhibitor may represent an effective strategy to eliminate differentiated TNBC cells and TNBC CICs.

Citation Format: Yangyang Wang, Francesco Sabbatino, Soldano Ferrone, Xinhui Wang. Inhibition of TNBC cell growth by CSPG4-specific mAb 225.28 with a Sonic Hedgehog pathway inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2013-4741
Abstract 4749: Cyclophosphamide induces dose dependent apoptosis of CD4+FoxP3+ regulatory T cells relative to CD4+FoxP3- effector T cells in breast cancer patients.
Authors: Chen Gang , Leatherman James M. , Sunay Melek Erdinc , Emens Leisha A.
Published: August 2014

CD4+FoxP3+ regulatory T cells (Tregs) accumulate at tumor sites and within the peripheral blood of patients with a variety of cancers. Tregs suppress antigen-specific immune responses, and represent a major barrier to the efficacy of cancer immunotherapy. We previously conducted a dose-ranging study of low dose Cyclophosphamide (0, 200, 250, and 350 mg/m2) given one day prior to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting HER-2+ breast cancer vaccine in 28 patients with metastatic breast cancer. We found that vaccine-induced HER-2-specific immunity was best optimized by 200 mg/m2 CY, and that doses of CY higher than 200 mg/m2 suppressed antigen-specific immunity. To further explore the pharmacodynamic impact of CY on immune priming, we analyzed Tregs and effector CD4+ T cells present in peripheral blood mononuclear cells (PBMC) collected pre- and post-vaccination with or without CY by flow cytometry. We found that the percentage of CD4+FoxP3+ Tregs in PBMC did not change with vaccination alone. Conversely, CY at doses of either 200 or 250 mg/m2 selectively induced the apoptosis of CD4+FoxP3+ Tregs relative to CD4+FoxP3− effector T cells, and CY at 350 mg/m2 induced apoptosis of both CD4+FoxP3+ Tregs and CD4+FoxP3− effector T cells. Finally, CY at all doses selectively decreased CCR7−CD45RA− effector memory Tregs and increased CCR7+CD45RA+ naïve Tregs relative to vaccine alone. Collectively, these data suggest that low dose CY selectively induces apoptosis of CD4+FoxP3+ Tregs relative to CD4+FoxP3− effector T cells, providing a window for more efficient induction of vaccine-induced HER-2-specific immunity.

Citation Format: Gang Chen, James M. Leatherman, Melek Erdinc Sunay, Leisha A. Emens. Cyclophosphamide induces dose dependent apoptosis of CD4+FoxP3+ regulatory T cells relative to CD4+FoxP3- effector T cells in breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4749. doi:10.1158/1538-7445.AM2013-4749
Abstract 4753: Ablation of N1-S1 hepatocellular carcinoma in rat livers with nanosecond pulsed electric fields and induction of protective immunity.
Authors: Chen Ru , Sain Nova , Harlow Tye , Shires Peter , Long Gary , Heller Ricahrd , Beebe Stephen
Published: August 2014

Nanosecond pulsed electric fields (nsPEFs) could be used to ablate local tumor masses through minimally invasive or open surgery delivery, but further pre-clinical studies are needed. NsPEFs not only induces permanent permeabilization of cells, but also impacts intracellular organelles and result in apoptosis and other forms of cell death in vitro and in vivo. Hepatocellular carcinoma (HCC) is the third most common cancer cause of mortality worldwide. A diagnosis of HCC carries a guarded prognosis, especially when metastasis is documented. Presently, reliable, curative treatments without recurrence for very early, early, and intermediate HCC stages are still in urgent need of attention. In these studies, we specifically test the efficacy of nsPEFs in an orthotopic HCC rat tumor model. Orthotropic insertion of stably transfected (mcherry and/or luciferase) N1-S1 cells in Sprague Dawley rat livers was used as a model for treating HCC. Cells were treated with pulses with 100 ns duration and electric field strengths of 50 kV/cm. Different pulse numbers were applied. Recent results suggest that treatments of 300 or 500 pulses were more effective when separated by a 6 minute interval than when applied as a continuous, uninterrupted single treatment. However over time and treatment of more than 20 animals, 1000 nanosecond pulses treatment consistently showed successful ablation by effectively inducing tumor cell death. A single treatment of 1000 nsPEFs successfully induces cell apoptosis by activating caspase-9, caspase-3, but not caspase-8 in some cells, indicating induction of intrinsic apoptosis mechanisms as well as caspase-independent cell death, like that observed in vitro with human Jurkat cells. More importantly, rats successfully treated with 1000 pulses showed protective responses to a second N1-S1 intra-hepatic challenge injection of N1-S1 cells, suggesting a host immune response, which is under exploration. As an ideal potential therapy for HCC, nsPEFs treatment did not show any damaging impact on liver blood flow. These studies demonstrate for the first time, successful intra-hepatic treatment of HCC with a potential induction of immunogenic cell death and a host immune response after nsPEF ablation.

Citation Format: Ru Chen, Nova Sain, Tye Harlow, Peter Shires, Gary Long, Ricahrd Heller, Stephen Beebe. Ablation of N1-S1 hepatocellular carcinoma in rat livers with nanosecond pulsed electric fields and induction of protective immunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4753. doi:10.1158/1538-7445.AM2013-4753
Abstract 4757: RARα1 as a novel drug target to improve conventional hormonal adjuvant therapy of breast cancer.
Authors: Chari Venkatesh V. , Trumbly Robert J. , d'A Salazar Marcela , Ratnam Manohar
Published: August 2014

Genetic or epigenetic changes must occur and be propagated for breast cancer cells to develop resistance to hormonal adjuvant therapies. This would require a basal level of cell cycling during hormone depletion or tamoxifen treatment. We have previously demonstrated that the retinoic acid receptor α1 (RARα1) apoprotein supports a basal level of proliferation in breast cancer cells that are either hormone depleted or treated with tamoxifen. This action of RARα1 was ligand-insensitive (reflecting a nonclassical mechanism) and involved the activation of growth supporting genes by the RARα1 apoprotein. As RARα1 is genetically redundant, we hypothesize that including targeted downregulation of RARα1 will improve the outcome of hormonal adjuvant therapy in breast cancer. Tissue array analysis indicated prevalence of RARα1 in clinical breast tumors covering a wide pathologic spectrum. We have synthesized a high affinity RARα-specific antagonist, AGN194301, as a means of selectively targeting RARα. AGN194301 did not affect the ability of RARα1 to support basal cell proliferation and did not alter the level of RARα1 protein. Preliminary SAR studies showed the positions at which the compound could be modified without abrogating its ability to bind to RARα. Further studies and strategies for attaining downregulation of RARα1 using modified versions of this compound will be discussed.

Citation Format: Venkatesh V. Chari, Robert J. Trumbly, Marcela d'A Salazar, Manohar Ratnam. RARα1 as a novel drug target to improve conventional hormonal adjuvant therapy of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4757. doi:10.1158/1538-7445.AM2013-4757
Abstract 4759: HOXB7 functions as a co-activator of estrogen receptor in the development of tamoxifen resistance.
Authors: Jin Kideok , Teo Wei Wen , Yoshida Takahiro , Park Sunju , Sukumar Saraswati
Published: August 2014

Background: Multiple factors including long term treatment with tamoxifen are involved in the development of selective estrogen receptor modulator (SERM)-resistance in ERα positive breast cancer. Increased expression of HER family members can also directly alter cellular responses to tamoxifen, but the mechanism underlying the increased expression of the HERs is not clear. In this report we show that HOXB7 is an upstream regulator of HER2 and ER expression.

Results: Overexpression of HOXB7 results in upregulation of HER2 and ERα target genes, while knockdown of HOXB7 with siRNA in tamoxifen-resistant cell lines causes decrease of HER2 and ERα target genes expression and loss of tamoxifen resistance. To mediate upregulation of HER2 and ER-target genes occurring in tamoxifen resistance, the HOXB7-ERα complex recruits ERα coactivators and promotes HER2- and ER-target genes transcriptional activity through direct binding to the HER2 enhancer and regulatory regions of ER target genes. However, depletion of HOXB7 negatively affects the binding affinity of its cofactors to the HER2 enhancer element and ER-binding sites. Also, it is likely that miR196a controls HOXB7 expression; reducing miR196a levels in tamoxifen-resistant cells causes an increase of HOXB7 expression. Overexpression of miR196a sensitizes MCF-7 tamoxifen resistant cells to tamoxifen through downregulation of HER2 and ER-target genes while inhibition of miR196a by anti-miR196a inhibitors increases tamoxifen resistance through the upregulation of HER and ER target genes in MCF-7 cells. Further, miRNA 196a is regulated by c-MYC which undergoes stabilization through the EGFR-HER2 signaling pathway. Depletion of c-MYC by MYC shRNA enhances tamoxifen sensitivity by increasing the level of miR196a transcripts. Also, by c-MYC ChIP assay, we found that c-MYC inhibits miR196a expression through direct binding to its promoter region.

Conclusions: Overexpression of HOXB7 is a key event in the initiation and maintenance of tamoxifen resistance. These studies suggest that HOXB7 acts as a key regulator orchestrating two major groups of target molecules, ERα and HER2, in the oncogene hierarchy. Blocking MYC expression or reexpression of miR196a in TAM-R cells may provide novel strategies for reversing SERM-resistance. Therefore, we have provided additional evidence that supports the thesis that functional antagonism of HOXB7 has the potential to circumvent tamoxifen resistance.

Citation Format: Kideok Jin, Wei Wen Teo, Takahiro Yoshida, Sunju Park, Saraswati Sukumar. HOXB7 functions as a co-activator of estrogen receptor in the development of tamoxifen resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4759. doi:10.1158/1538-7445.AM2013-4759
Abstract 4761: Glial cell derived neurotrophic factor (GDNF)-RET signaling as a target in aromatase inhibitor resistant ER-positive breast cancers.
Authors: Morandi Andrea , Martin Lesley-Ann , Gao Qiong , Mackay Alan , Robertson David , Zvelebil Marketa , Dowsett Mitch , Plaza-Menacho Ivan , Isacke Clare M.
Published: August 2014

The majority of breast cancers are estrogen receptor (ER)-positive and depend on estrogen for growth and survival. Blockade of estrogen biosynthesis by aromatase inhibitors (AIs) is the first-line endocrine therapy for post-menopausal women with ER-positive breast cancers. Despite providing substantial improvements in patient outcome, resistance to such therapy remains a major clinical challenge. We previously showed that GDNF-RET signaling plays an important role in ER-positive breast tumors and in the response to tamoxifen treatment. Resistance to this therapy is often associated with estrogen-independent activation of ER mediated by cell surface growth factor receptors, such as RET. Here, a multidisciplinary strategy was used to address the impact of GDNF-RET signaling in the response to AI treatment. First, using 2D and 3D in vitro approaches we show that GDNF-mediated RET signaling is enhanced in a model of AI resistance. Further, GDNF-RET signaling promotes the survival of AI-resistant cells and increases resistance in AI-sensitive cells, effects that are selectively reverted by a potent RET kinase inhibitor.

Second, we generated a proliferation-independent GDNF-response gene set, identified from gene expression profiling in ER-positive breast cancer cells, and demonstrated this to be an independent prognostic marker of poor patient outcome and, importantly, to be predictive of poor response to AI treatment and development of resistance. Finally, the relevance of these findings was validated by demonstrating increased RET protein expression levels in an independent cohort of AI resistant patient samples. Together these preclinical and clinical models identify GDNF-RET signaling as a key determinant of response and resistance to AIs in ER-positive breast cancers. As a consequence, RET inhibition may be of use in the clinical setting either in combination with an AI to prevent and delay the onset of resistance, or to prolong the efficacy of the AI in the recurrent and/or metastatic setting.

Citation Format: Andrea Morandi, Lesley-Ann Martin, Qiong Gao, Alan Mackay, David Robertson, Marketa Zvelebil, Mitch Dowsett, Ivan Plaza-Menacho, Clare M. Isacke. Glial cell derived neurotrophic factor (GDNF)-RET signaling as a target in aromatase inhibitor resistant ER-positive breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4761. doi:10.1158/1538-7445.AM2013-4761
Abstract 4766: Follicle stimulating hormone (FSH) inhibits apoptosis in ovarian cancer cell through regulating OCT4 stem cell signal pathway.
Authors: Zhang Zhenbo , Zhu Yaping , Xu Xianming , Xi Xiaowei , Bast Robert C. , Yu Yinhua , Feng Youji
Published: August 2014

Ovarian cancer is the most lethal malignancy of the female reproductive system with an overall mortality rate of greater than 70%. Our group has demonstrated that Follicle stimulating hormone (FSH) stimulates the proliferation and invasion of ovarian cancer cells, inhibits apoptosis, and facilitates neovascularization by increasing expression of vascular endothelial growth factor (VEGF). OCT4, a stem cell marker, is overexpressed in several types of human cancer and can induce resistance to chemotherapy and inhibition of apoptosis. We have found that FSH stimulates the expression of OCT4 mRNA and protein in a time- and dose-dependent manner. Subcellular analysis by immunofluorescent staining confirmed that FSH stimulation increased OCT4 protein expression in both the cytoplasm and nucleus of ovarian cancer cells. Re-expression of OCT4 in ovarian cancer cells reversed the inhibition of apoptosis induced by FSH, whereas siRNA-mediated knockdown of OCT4 resulted in a significantly rate of apoptosis. Depletion of OCT4 resulted in reduction of p-AKT and survivin, and also blocked FSH-induced upregulation of p-AKT and survivin. Moreover, FSH stimulation induced the expansion of CD44+CD117+ cells with a stem cell-like phenotype. Interestingly, re-expression of OCT4 enhanced the expression of Notch, Sox2, and Nanog, molecules that play critical roles in cancer stem cell proliferation and differentiation. FSH upregulated the expression of Notch, Sox2 and Nanog and these effects were abolished by knocking down OCT4, suggesting that several cancer stem cell pathways are involved in FSH regulation. We have also examined OCT4 expression in surgical specimens of ovarian cancer. Immunohistostaining revealed that OCT4 expression was increased in ovarian carcinoma compared with benign cystadenomas and borderline tumors, and OCT4 expression was significantly correlated with histological grade. Staining for OCT4 was increased in serous cystadenocarcinoma, when compared with clear cell carcinoma. In summary, the OCT4 cancer stem cell signaling pathway may mediate FSH-induced inhibition of apoptosis and could provide a target for treatment of ovarian cancer.

Citation Format: Zhenbo Zhang, Yaping Zhu, Xianming Xu, Xiaowei Xi, Robert C. Bast, Yinhua Yu, Youji Feng. Follicle stimulating hormone (FSH) inhibits apoptosis in ovarian cancer cell through regulating OCT4 stem cell signal pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4766. doi:10.1158/1538-7445.AM2013-4766
Abstract 4789: Human papillomavirus genotyping in urine samples of women with squamous intraepithelial lesions of cervix.
Authors: Viscarra Tamara , Andana Alejandra , Brebi Priscilla , Menzel Doris , Ili Carmen G. , Silva Ramon , Roa Juan C. , Sanchez Raul
Published: August 2014

Human papillomavirus (HPV) is the most frequent microorganism sexually transmitted and is the causal agent of cervical carcinoma were it could be found in 99.7% of cases. The detection of HPV en women with or without lesions of cervix is generally performed in biopsies and cytobrush, which are invasive samples; therefore many patients do not attend to health care to get HPV test. The aim of this study was to develop a technique capable of detect and genotype HPV in urine samples of women with squamous intraepithelial lesions of cervix.

For this study, 87 urine samples from patients with normal epithelia of cervix (25), low grade squamous intraepithelial lesion (LSIL) (24), high grade squamous intraepithelial lesion (HSIL) (38) were collected from women who attended to gynecological care in a public health center in Temuco, Chile. DNA extraction was performed with a manual method. For protein precipitation, Phenol-Chloroform and ammonium acetate were used. DNA precipitation was performed with absolute ethanol. HPV detection and genotyping was performed with the kit HPV Typing (patent pending) which is capable of detect 18 more frequent HPV types and beta globin gen as control of DNA integrity.

20% of urines from normal epithelia women were positive for HPV. Three genotypes were found in normal samples: HPV52, HPV42 and HPV53. LSIL there was a 92% of HPV positivity and the most frequent genotypes were HPV16 and HPV18. In HSIL, 95% of urine sample were positive for HPV. HPV16, HPV18 and HPV31 were the most frequent genotypes in HSIL. Genotypes found in urine samples were similar to world reports.

Using the DNA extraction described before and HPV easy typing kit it is possible to detect and genotype HPV in urine samples. These results suggest that is possible a HPV primary screening test in non-invasive sample, complementing PAP smear in order to determinate which women have a higher risk of developing preneoplastic and neoplastics lesions.

Grant Support: Proyect CORFO N° 09CN14-5960 (CEGIN). CI is recipient of grants from FONDECYT Postdoctoral Proyect N° 3130630. PB is recipient of grants from FONDECYT Postdoctoral Proyect N° 3120141.

Citation Format: Tamara Viscarra, Alejandra Andana, Priscilla Brebi, Doris Menzel, Carmen G. Ili, Ramon Silva, Juan C. Roa, Raul Sanchez. Human papillomavirus genotyping in urine samples of women with squamous intraepithelial lesions of cervix. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4789. doi:10.1158/1538-7445.AM2013-4789
Abstract 4792: Analysis of the relationship between experimentally induced rodent forestomach hyperplasia and tumorigenesis.
Authors: Fritz Jason M. , Gwinn Maureen R.
Published: August 2014

Hyperplasia is an increase in cell number resulting from physiological processes such as irritation, or pathological processes like carcinogenesis. The difficulty in differentiating reactive versus preneoplastic (or dedicated) hyperplasia has been the subject of debate in the literature, especially in regard to prediction of human health risks following chronic chemical exposure. Squamous hyperplasia and neoplasia are frequently detected in the rodent forestomach (FS) following chronic oral administration of chemicals evaluated in rodent cancer bioassays. Molecular level mechanistic details related to FS hyperplasia progression are not available for a wide range of chemicals, despite the increasing need for this level of mechanistic understanding to support evaluations for human health risk. In order to facilitate further discussion on the use of rodent FS lesions in the estimation of cancer and noncancer reference values, a comparison of chemical hyperplasia versus tumor induction was performed. FS lesion classification and incidence data were obtained from rat and mouse gavage bioassays on over 50 chemicals from National Toxicology Program (NTP) 2-year bioassays that reported dose-related increases in FS hyperplastic or neoplastic lesions in one or more rodent sex/strain combinations tested. Chemical effects were tabulated and sorted by strain, sex, genotoxicity assay results and irritation effects, and the relationship between FS hyperplasia and FS tumor (e.g., squamous cell papilloma or carcinoma) induction was analyzed using grouped animal data. When considering chemicals with positive results in genotoxicity assays, FS tumors positively correlated with FS hyperplasia incidence in both rats and mice. Increased incidence of FS hyperplasia was not correlated with FS tumors in animals exposed to chemicals that were negative for genotoxicity and irritation. The potential results of this analysis may be useful in the evaluation of chemicals with limited chronic bioassay data by informing predictions of long-term tumor formation based on chemical properties observable during short-term animal or in vitro studies (e.g., genotoxicity, irritation).

The views expressed in this abstract are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency.

Citation Format: Jason M. Fritz, Maureen R. Gwinn. Analysis of the relationship between experimentally induced rodent forestomach hyperplasia and tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4792. doi:10.1158/1538-7445.AM2013-4792
Abstract 4794: An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through upregulation of CXCR4.
Authors: Arora Sumit , Bhardwaj Arun , Singh Seema , Srivastava Sanjeev K. , McClellan Steven , Grizzle William E. , Owen Laurie B. , Singh Ajay P.
Published: August 2014

Pancreatic cancer (PC) remains a therapeutic challenge with a rising incidence and unabated mortality. Gemcitabine (GEM), the current standard of care drug, is largely ineffective due to chemoresistance, thus underscoring the need for a refined understanding of the molecular mechanisms that drive drug-resistant nature of this malignancy. Recently, we have shown that CXCL12/CXCR4 signaling axis is involved in conferring chemoresistance to PC cells. Here, we explored the effect of GEM on this resistance mechanism and its functional consequences. Our data demonstrated that GEM upregulated CXCR4 expression in two PC cell lines (MiaPaCa and Colo357) in a dose- and time- dependent manner. GEM-induced CXCR4 expression was dependent on generation of reactive oxygen species (ROS) as it was abrogated upon pre-treatment of PC cells with radical scavenger N-acetyl-L-cysteine (NAC). CXCR4 upregulation by GEM correlated with time-dependent accumulation of p65/NF-κB and HIF-1α in the nucleus. An enhanced binding of p65/NF-κB and HIF-1α to the CXCR4 promoter was observed in chromatin immunoprecipitation assay in GEM-treated PC cells, while their silencing by RNA interference suppressed GEM-induced CXCR4 expression. ROS induction upon GEM treatment preceded the nuclear accumulation of p65/NF-κB and HIF-1α and suppression of ROS diminished these effects. Effect of ROS on p65/NF-κB and HIF-1α was mediated through activation of ERK1/2 and Akt and their pharmacological inhibition also suppressed GEM-induced CXCR4 upregulation. Lastly, our data demonstrated that GEM-treated PC cells were more motile and exhibited significantly greater invasiveness against a CXCL12 gradient. Altogether, these findings reinforces the role of CXCL12/CXCR4 signaling in GEM resistance and point toward an undesired effect of chemotherapy that is potentiation of invasion and spread of resistant tumor cells.

Citation Format: Sumit Arora, Arun Bhardwaj, Seema Singh, Sanjeev K. Srivastava, Steven McClellan, William E. Grizzle, Laurie B. Owen, Ajay P. Singh. An undesired effect of chemotherapy: gemcitabine promotes pancreatic cancer cell invasiveness through upregulation of CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2013-4794
Abstract 4801: Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner.
Authors: Hill Thomas , Alexander Carla-Maria , Bauer Alison K.
Published: August 2014

Subchronic dosing of BHT in mice is a well-known model used to discretely study the tumor promotion stage of lung adenocarcinoma. Using this model, we have previously shown that functional toll-like receptor 4 (TLR4) is protective against inflammatory sequelae and primary tumor formation in the mouse lung. Gap junctional intercellular communication (GJIC) has been shown to regulate the signal transduction pathways of cells, and inhibition of this communication by toxicant exposure has been linked to dysregulation and eventual carcinogenic endpoints. As part of our research on the protective nature of TLR4 during tumor promotion, we utilized the BHT subchronic model (4 weekly i.p. doses of BHT) to examine the early effects on BALF in both BALB/c (BALB; Tlr4 sufficient) and C.C3H-Tlr4Lps-d mice (BALBLps-d-d; Tlr4 mutant). Analysis of the BALF 24 hours after the final dose for protein content and cytology was indicative of an inflammatory profile in all mice treated with BHT. Magnitude of response to BHT was highest in the BALBLps-d mice; the BALB appeared resistant. In an effort to further characterize the response of the pulmonary epithelium, the BALF samples were used to treat cultured C10 cells, a murine immortalized line derived from BALB/c alveolar type II cells, which are a precursor cell type for adenocarcinoma. Using the scrape-load dye transfer assay, a well-defined method to measure GJIC, we demonstrated marked inhibition of cellular communication after a 4 hour exposure to cell-free BALF that is both treatment and strain dependent. The direction and magnitude of effect is in parallel with that of the direct analysis of the BALF, i.e. we observed more inhibition in BALF from BALBLps-d compared to BALB mice. In addition, quantitative PCR (QPCR) analysis of C10 cells exposed to cell-free BALF for 24 hours indicates alterations in the transcription patterns in genes of innate immunity that are also treatment and strain dependent. These novel findings suggest endocrine signaling from the inflammatory milieu are complicit in the inhibition of GJIC during the promotion phase of carcinogenesis. Furthermore, these data establish a link between active gap junctions, tumor promotion, and the protective nature of TLR4 in an ex vivo model which will facilitate a mechanistic examination of these protective effects at the molecular level. Funded by ACS RSG-10-162-01-LIB (AKB).

Citation Format: Thomas Hill, Carla-Maria Alexander, Alison K. Bauer. Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2013-4801
Abstract 4818: Type 2 diabetes mellitus and colorectal neoplasia risk in Puerto Rican Hispanics: A case-control study.
Authors: Diaz-Algorri Yaritza , Gonzalez Dilka , Lozada Maria E. , Lopez-Diaz Sofia Margarita , Hernandez Jessica , Pedrosa Carmen , Toro Doris , Criuz-Correa Marcia R.
Published: August 2014

Introduction: Epidemiological studies have provided inconclusive evidence regarding the potential link between type 2 diabetes mellitus (DM) and colorectal cancer (CRC).The association between type 2 DM and colorectal neoplasia (colorectal cancer and/or adenomas) has not been well studied in Hispanics, an ethnic minority at high risk for type 2 DM.The specific aim of the study was to evaluate the association between type 2 DM and colorectal neoplasia (CRN) in Puerto Rican Hispanic adults enrolled in the studies Epidemiology of Loss of Imprinting in Colorectal Cancer, Familial Colorectal Cancer Registry and the Type 2 diabetes mellitus and colorectal neoplasia risk in Hispanics: A case-control study at the VA Caribbean Healthcare System. Methods: The case-control study included patients with incident CRN and controls with negative colonoscopy and without previous history of CRC or adenomas diagnosed from January 1, 2005 to December 31, 2009. Diagnosis of type 2 DM was established by previous medical diagnosis and/or anti-diabetic medications use. Unconditional logistic regression was employed to estimate the odds ratio (OR) between type 2 DM and CRN using STATA 10.0. Results: A total of 501 participants (mean age 60.3 ± 12.6 yrs., 57.9% males), prevalence of type 2 DM was 24.8%. 323 patients with CRN and 178 controls were evaluated. In the cases, the prevalence of colorectal adenomas was 25.7% (83/323) and the prevalence of colorectal cancer was 74.3% (240/323). Cases and controls did not differ by gender (p=0.19), median age (p=0.91), family history of CRC (p=0.76) and first degree of family history of DM (p=0.49). Cases had lower education (p<0.001) and reported lower use of NSAIDS (p=0.054) and physical activity (p=0.005) compared to the controls. There were no statistical significant associations between DM and CRN (OR=1.21; 95% CI: 0.79-1.87), DM and CRC (OR=1.20; 95% CI: 0.76-1.90) or DM and colorectal adenomas (OR=1.24; 95% CI: 0.68-2.27). Conclusions: We did not observe a statistical significant association between type 2 DM and CRN. Nonetheless, a tendency towards an increase risk of colorectal neoplasia was observed among type 2 DM patients. Possible explanation for our lack of association may be related to high prevalence of type 2 DM in cases and controls.

Citation Format: Yaritza Diaz-Algorri, Dilka Gonzalez, Maria E. Lozada, Sofia Margarita Lopez-Diaz, Jessica Hernandez, Carmen Pedrosa, Doris Toro, Marcia R. Criuz-Correa. Type 2 diabetes mellitus and colorectal neoplasia risk in Puerto Rican Hispanics: A case-control study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4818. doi:10.1158/1538-7445.AM2013-4818
Abstract 4820: Habitual intake of total flavonoids and flavonoid subclasses and risk of colorectal cancer in men and women.
Authors: Nimptsch Katharina , Zhang Xuehong , Giovannucci Edward , Cassidy Aedin , O'Reilly Éilis J. , Pischon Tobias , Rimm Eric B. , Lin Jennifer , Willett Walter C. , Fuchs Charles S. , Wu Kana
Published: August 2014

Background: Flavonoids inhibit the growth of colon cancer cells in vitro. In a recent randomized controlled trial, the Polyp Prevention Trial, a higher intake of one sub-class, flavonols, was significantly associated with a 76% reduced risk of recurrent advanced adenoma. Cohort studies that have examined the association between flavonoid intake and risk of colorectal cancer (CRC) have yielded inconsistent results, but most previous studies only evaluated habitual intake of a limited number of subclasses and there is growing evidence for differential effects depending on subtle difference in structure between the sub-classes

Objective: To examine whether higher habitual dietary intake of total flavonoids and their subclasses (flavanones, anthocyanins, flavan-3-ols, flavonols, flavones and polymers) are associated with lower risk of CRC.

Methods: Using data from validated food frequency questionnaires administered every four years and an updated USDA flavonoid food composition database flavonoid intakes were calculated for 73,666 participants from the Nurses’ Health Study I, and for 32,059 participants from the Health Professionals Follow-up Study.

Results: During 16 years of follow-up, 1,405 colorectal cancer cases (652 in men, 753 in women) were identified. Intakes of total flavonoids and subclasses were not associated with risk of CRC (hazard ratios (95% confidence interval) multivariable adjusted for age, smoking before age 30, family history of colorectal cancer, history of endoscopy, aspirin use, body mass index, physical activity, postmenopausal hormone use in women, alcohol consumption, intake of total energy, calcium and vitamin D; highest vs. lowest quintile: total flavonoids, men: 0.97 (0.75-1.26), women: 1.00 (0.80-1.26); flavonols, men: 0.98 (0.76-1.26), women: 1.21 (0.96-1.52); flavones, men: 0.86 (0.67-1.11), women: 0.90 (0.72-1.13); flavanones, men: 0.98 (0.75-1.28), women: 1.00 (0.80-1.26); flavan-3-ols, men: 1.00 (0.78-1.29), women: 0.96 (0.76-1.21); anthocyanins, men: 0.89 (0.69-1.15), women: 1.25 (0.98-1.60); polymers, men: 0.92 (0.71-1.18), women: 0.88 (0.69-1.11)).

Conclusion: Our findings do not provide support for the hypothesis that a higher intake of flavonoids or any specific sub-class decrease risk of CRC.

Citation Format: Katharina Nimptsch, Xuehong Zhang, Edward Giovannucci, Aedin Cassidy, Éilis J. O'Reilly, Tobias Pischon, Eric B. Rimm, Jennifer Lin, Walter C. Willett, Charles S. Fuchs, Kana Wu. Habitual intake of total flavonoids and flavonoid subclasses and risk of colorectal cancer in men and women. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4820. doi:10.1158/1538-7445.AM2013-4820
Abstract 4866: CLEFMA induces apoptosis and downregulates inflammatory markers in lung cancer model.
Authors: Yadav Vivek R. , Sahoo Kaustuv , Awasthi Vibhudutta
Published: August 2014

Background: Inflammation is a precursor for many cancers. NF-kB plays an important role in inflammation, is associated with cancerous growth and has been identified as a potential target for development of anticancer therapies. Lung cancer is a classic example of inflammation-induced carcinogenesis where it prevents apoptosis and stimulates production of pro-inflammatory cytokines. CLEFMA or 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] is a new molecule that potently inhibits proliferation of various cancer cells without affecting normal cells. In this study, we evaluated the antiproliferative activity of CLEFMA in a mouse xenograft model of human lung cancer.

Methods: Cell viability was determined in human lung cancer cells H441 and A549 after treatment with CLEFMA. Immunobloting studies were performed in cell lysates and tissue homogenates for anti-apoptotic and pro-apoptotic markers. These were further confirmed by immunohistochemical studies. The ability of CLEFMA in reducing the metabolic turnover in tumor tissue was examined by positron emission tomography using F-18-FDG as a biomarker of glucose metabolism.

Results: We found that CLEFMA-induced suppression of lung adenocarcinoma H441 and A549 cell proliferation was associated with cleavage of caspases 3/9 and PARP, suggesting apoptotic cell death. In vivo, CLEFMA demonstrated a dose-dependent suppression of tumor growth associated with reduction of tumor proliferation marker Ki-67. Non-invasive PET imaging with F-18-flurodeoxyglucose also showed abrogation of glucose uptake in tumor tissue. In the tumor tissue homogenates, we found inhibition of anti-apoptotic markers cIAP1, Bcl-XL, BCL2 and survivin, and upregulation of pro-apoptotic BID cleavage, BAX and PARP. Further, CLEFMA inhibited NF-kB at mRNA level and reduced phospho-p65 translocation into the nucleus. It also reduced the expression of COX-2 in tumor tissue, and significantly reduced serum pro-inflammatory cytokines TNF-α and IL-6.

Conclusions: The findings suggest that CLEFMA inhibits growth of lung cancer xenografts by activating caspase-dependent apoptotic pathway, and the tumor suppression is associated with anti-inflammatory and anti-metastatic effects, perhaps mediated by NF-kB.

Citation Format: Vivek R. Yadav, Kaustuv Sahoo, Vibhudutta Awasthi. CLEFMA induces apoptosis and downregulates inflammatory markers in lung cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4866. doi:10.1158/1538-7445.AM2013-4866
Abstract 4869: β-caryophyllene inhibits solid tumor growth and metastasis of B16F10 melanoma cells in C57BL/6 mice fed on a high-fat diet.
Authors: Jung Jae In , Kim Eun Ji , Jung Yoo Jin , Chun Hyang Sook , Park Jung Han Yoon
Published: August 2014

We previously observed that chronic consumption of a high-fat diet (HFD) increased the melanoma progression and lymph node metastasis in C57BL/6N mice injected with B16F10 melanoma cells. β-caryophyllene (BCP) is a natural bicyclic sesquiterpene found in many essential oils especially clove and oregano oils. In the present study, we examined whether BCP inhibits HFD-induced melanoma growth and metastasis in the B16F10 allograft model. Four-week old, male C57BL/6 mice were fed on a HFD (60 kcal% fat) or control diet (CD, 10 kcal% fat) with or without BCP (0.15, 0.3% in diet) for a period of 21 weeks. Following 16 weeks of either CD or HFD in the absence or presence of BCP, B16F10 melanoma cells were subcutaneously injected into mice and all mice were continuously fed identical diets. Three weeks post-injection, tumors were resected and mice were sacrificed three weeks post-resection. The body weights and the growth of solid tumors were markedly increased in the HFD group as compared to the CD group, and these increases were suppressed by BCP treatment. The expression of proteins related to cell proliferation (Ki67, CDK4, cyclin A, and cyclin D1), angiogenesis (VEGF-A, VEGFR-2, and CD31) and lymphangiogenesis (VEGF-C, VEGF-D, VEGFR-3, and LYVE-1) was markedly increased in tumor tissues of the HFD group as compared to the CD group, and these increases were blocked by BCP treatment. HFD feeding increased the infiltration of CD45+ leukocytes and F4/80+ macrophages into tumor tissues. The administration of BCP significantly suppressed HFD-induced the infiltration of the immune cells into tumor tissues. HFD feeding reduced TUNEL-positive apoptotic cells in the tumor tissues, which was prevented by BCP treatment. Two weeks after tumor resection, the mice evidenced lung and lymph node metastasis. Lung and lymph node metastasis were increased in the mice fed on HFD, and these increases were markedly suppressed by BCP treatment. The present results demonstrate that BCP suppressed HFD-stimulated melanoma progression and metastasis, which may have been mediated via the suppression of cell cycle progression, angiogenesis and lymph angiogenesis, and induction of apoptosis in tumor tissues. These results suggest that BCP has potential as an anti-cancer agent for the prevention of obesity-stimulated melanoma progression.

Citation Format: Jae In Jung, Eun Ji Kim, Yoo Jin Jung, Hyang Sook Chun, Jung Han Yoon Park. β-caryophyllene inhibits solid tumor growth and metastasis of B16F10 melanoma cells in C57BL/6 mice fed on a high-fat diet. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4869. doi:10.1158/1538-7445.AM2013-4869