bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4044: Effect of adjuvant chemotherapy on the phenotype of circulating tumor cells in patients with triple negative breast cancer
Authors: Kallergi Galatea , Dragolia Melina , Mavroudis Dimitris , Georgoulias Vassilis , Agelaki Sofia
Published: May 2015

Introduction: Recent studies revealed phenotypic discrepancies between Circulating Tumor Cells' phenotype (CTCs) and the corresponding primary tumor in patients with breast cancer. Patients with triple negative (TN) (ER-negative, PR-negative, HER2-negative) primary tumors are considered as a poor prognosis group for whom no targeted therapies are currently available. The aim of the current study was to characterize CTCs in TN breast cancer (BC) patients before and after adjuvant chemotherapy in order to explore additional therapeutic approaches based on the phenotype of chemoresistant CTCs.

Methods: Cytospins were prepared from peripheral blood mononuclear cells (PBMC), obtained by ficoll density gradient centrifugation of 31 TN BC patients, before and after adjuvant treatment. The expression of Cytokeratins (CK), Estrogen Receptor (ER), Progesterone Receptor (PR), EGFR and HER2 in CTCs was assessed using double immunofluorescent experiments and ARIOL analysis.

Results: CTCs were detected in 25 out of 31 (81%) patients before and in 18 out of 31 (58%) after adjuvant chemotherapy. ER, PR, EGFR and HER2 expression was detected in 33.7%, 23.0%, 69.3%, 42.8% of the total examined CTCs before adjuvant chemotherapy and in 4.2%, 0%, 61.79% and 15.73% after treatment. The frequency of ER- and PR- expressing CTCs was significantly (p=0.05, p=0.007) reduced post-chemotherapy. The absolute number of ER and PR positive-CTCs per patient was also statistically decreased (p=0.02, p=0.001) Triple staining experiments could not reveal co-expression of CK/EGFR/ER in CTCs; however, there was one patient with CTCs co-expressing CK/HER2/ER.

Conclusions: A significant percentage of CTCs in triple negative breast cancer patients express HER2 and EGFR before treatment, implying that these receptors could be a potential target for the limitation of metastasis in these patients. EGFR-expressing CTCs persist after adjuvant chemotherapy, suggesting that additional treatment with EGFR-targeting agents could be used post-chemotherapy to eliminate chemo-resistant CTCs.

Citation Format: Galatea Kallergi, Melina Dragolia, Dimitris Mavroudis, Vassilis Georgoulias, Sofia Agelaki. Effect of adjuvant chemotherapy on the phenotype of circulating tumor cells in patients with triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4044. doi:10.1158/1538-7445.AM2014-4044
Abstract 4047: Potential role of CXCR6-CXCL16 in prostate cancer progression and chemotherapeutic efficacy
Authors: Gupta Pranav , Ward Ashley B. , Mir Hina , Kaur Gurpreet , Grizzle William E. , Lillard James W. , Singh Shailesh
Published: May 2015

Despite recent advances in treatment and clinical management, prostate cancer is still a leading cause of cancer related deaths among men, primarily because etiopathogenesis of PCa is not well defined. In this regard, chemokines and their corresponding receptors have been shown to play major role in PCa progression, which are often associated with poor therapeutic outcomes. Hence, the focus of this study is to determine the potential role of CXCR6 and its natural ligand CXCL16 in PCa pathogenesis, and potential impact of CXCR6-CXCL16 axis on efficacy of docetaxel, which is currently offered in the clinics as standard care.

We investigated expression of CXCR6 and CXCL16 in clinical samples, prostate cancer cell lines and normal prostatic epithelial cells. Expression of CXCR6 and CXCL16 was significantly higher in PCa samples compared to their respective controls, and expression of CXCR6/ CXCL16 was correlated with tumor stage and grades. Similar to patient's samples, expression of CXCR6/CXCL16 was significantly higher in PCa cell lines compared to normal prostatic epithelial cells. Furthermore, levels of phospho -ERK1/2 and -NF-kB, known to be involved in cell growth and survival, were significantly higher in PCa cells treated with CXCL16 compared to untreated controls. In addition to these, role of CXCR6- CXCL16 on genes responsible for cellular adhesion and epithelial-mesenchymal transition were examined. Interestingly, expression of surface E-cadherin and β−catenin following CXCL16 treatment was significantly inhibited compared to untreated cells, suggesting the involvement of CXCR6-CXCL16 interaction in PCa cell migration. Additionally, CXCR6 expressing PCa cells expressed higher α−smooth muscle actin protein, another EMT marker presumably induced by elevated TGF-β, following CXCL16 stimulation. Furthermore, expression of CXCR6, CXCL16 and MMP(s) were elevated in PCa cell following docetaxel treatment in a time and dose dependent manner compared to controls. This may explain the increased efficacy of docetaxel when used in combination with anti-CXCR6 antibody as compared to docetaxel alone. These findings suggest that CXCR6-CXCL16 axis plays a crucial role in pathogenesis of PCa, and inhibition of this axis may have significant impact on disease progression and therapeutics outcomes.

Citation Format: Pranav Gupta, Ashley B. Ward, Hina Mir, Gurpreet Kaur, William E. Grizzle, James W. Lillard, Shailesh Singh. Potential role of CXCR6-CXCL16 in prostate cancer progression and chemotherapeutic efficacy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4047. doi:10.1158/1538-7445.AM2014-4047
Abstract 4055: The transcriptional regulator TBX3 promotes progression of cells representing early premalignant breast cancer
Authors: MacMillan Connor D. , Leong Hon S. , Clifford Allen G. , Krstic Milica , Pardhan Siddika , Dales David W. , Postenka Carl O. , Chambers Ann F. , Tuck Alan B.
Published: May 2015

The transcriptional regulator TBX3 has been shown to be elevated in metastatic breast cancer and is thought to be involved in promoting malignancy of breast and other carcinomas. In a series of cell lines derived from the same patient with breast cancer, we previously showed that metastatic 21MT-1 cells express elevated levels of TBX3, while low levels are expressed by the ductal carcinoma in situ-like 21NT cells. Here we hypothesized that upregulation of TBX3 in 21NT cells would promote their progression to a more malignant phenotype. We found that 21NT cells engineered to overexpress either isoform of TBX3 (iso1, iso2) have an altered growth in 3D Matrigel, with increased colony-forming ability, higher number of cells per colony and less spherical, more irregular colonies, consistent with increased invasiveness. Transwell assays through Matrigel showed increased invasiveness of 21NT cells overexpressing either TBX3 isoform and in vivo assay in the chick embryo similarly showed increased propensity of TBX3 over-expressing 21NT cells for extravasation. These TBX3-induced changes were associated with increased vimentin expression, but minimal change in e-cadherin expression. Overall, these results indicate that TBX3 (iso1 or iso2) expression can promote progression in a model of early breast cancer, by altering cell properties involved in cell survival/colony formation and invasiveness.

Citation Format: Connor D. MacMillan, Hon S. Leong, Allen G. Clifford, Milica Krstic, Siddika Pardhan, David W. Dales, Carl O. Postenka, Ann F. Chambers, Alan B. Tuck. The transcriptional regulator TBX3 promotes progression of cells representing early premalignant breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4055. doi:10.1158/1538-7445.AM2014-4055
Abstract 4057: Modulation of actin binding proteins by docosahexaenoic acid reduces cancer cell migration
Authors: Ali Mehboob , Long Alexander , Heyob Kathryn M. , Srinivasan Asha , Rogers Lynette K.
Published: May 2015

Cancer is the leading cause of death worldwide and all age groups, including children, are at risk for cancer associated death. Disease progression and metastasis are major contributors to cancer-associated morbidity and mortality. Increased cell migration rate is characteristic of tumor progression and metastasis. Actin binding proteins regulate cytoskeletal remodeling at the leading edges of cancer cells, facilitate invasive organelle (invadopodia) formation, and promote higher migration rates. Docosahexaenoic acid (DHA), a poly unsaturated fatty acid, has been shown to inhibit the cancer cell metastatic phenotype. Here, we test the hypothesis that changes in actin binding proteins regulate cancer cell migration and that supplementing cells with DHA will prevent these changes. Non-cancer (MLE12) and cancer (A549, HCT116 and MCF7) cells were treated with 8-Br-cAMP and/or DHA. F-actin content was measured using confocal microscopy. Cell migration was estimated by wound assay and transwell apparatus. Actin binding proteins, profilin, cofilin, vimentin and gelsolin, were identified and quantified using confocal microscopy and western blot to evaluate wound edges and actin co- munoprecipitate, respectively. F-actin content and cell migration were increased by cAMP in association with changes in profilin, cofilin, vimentin and gelsolin levels. DHA treatment suppressed the increase in actin content and cell migration in cancer cells but not in non-cancer cells in association with altered levels of actin binding proteins. We postulate that DHA specifically inhibits cancer cell migration via alterations in actin binding proteins indicating a therapeutic potential against cancer cell metastasis. The changes in actin binding proteins could serve as biomarkers for cancer progression and as innovative therapeutic targets.

Citation Format: Mehboob Ali, Alexander Long, Kathryn M. Heyob, Asha Srinivasan, Lynette K. Rogers. Modulation of actin binding proteins by docosahexaenoic acid reduces cancer cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4057. doi:10.1158/1538-7445.AM2014-4057
Abstract 406: A potential new mechanism for PTEN to maintain genome stability
Authors: Gong Lili , Lin Shiaw-Yih
Published: May 2015

Purpose: PTEN (phosphatase and tensin homolog) is among the most mutated or lost tumor suppressors. Recent studies revealed that nuclear PTEN represses tumorigenesis by stabilizing the centromere, a highly condensed heterochromatin domain featured with long array of DNA repeats (namely, satellite DNA), repressive histones, such as trimethylated Histone H3 at Lys9 residue (H3K9me3), and heterochromatin proteins. Our studies are aimed to explore the molecular mechanism that PTEN regulates this heterochromatin structure and thus maintaining genome stability.

Methods and Results: Co-immunoprecipitation (Co-IP) assay showed that PTEN and the heterochromatin proteins directly interact. Chromatin immunoprecipitation (ChIP) assay showed that PTEN is enriched in satellite locus. Depletion of PTEN leads to abnormal heterochromatin structure, indicated by reduced H3K9m3 foci (detected by immunofluorescent staining) and occupancy to satellite locus (ChIP assay), decreased heterochromatin protein levels (Westernblot analysis), and overexpression of satellite transcripts (quantitative RT-PCR). Through a knockdown-and-mutant rescue strategy, we found that the C-terminal of PTEN is essential for its repressive function on satellite transcription. Furthermore, overexpression of the C-terminal truncated PTEN into PTEN-null BT549 cells, as compared to wild-type PTEN overexpression, lost its function to suppress cell growth (MTT assay), cell cycle progression (flow cytometry analysis) and protection from DNA damage (MTT and colony formation assays).

Conclusions: Our finding showed that PTEN guards the genome stability through maintaining the normal structure of heterochromatin domain.

Citation Format: Lili Gong, Shiaw-Yih Lin. A potential new mechanism for PTEN to maintain genome stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2014-406
Abstract 4063: Potential targets of PTP4A3 involved in uveal melanoma migration
Authors: Liot Geraldine , Anezo Oceane , Laurent Cecile , Maacha Selma , Planque Nathalie , Saule Simon
Published: May 2015

Uveal melanoma (UM) is the most frequent eye cancer in the adulthood and it represents 4-5% of all melanomas. Although at diagnosis over 95% of patients have disease limited to the eye, about 50% will develop metastases after a median time of three years. From transcriptomic study, we identified PTP4A3/PRL3 level as an efficient predictor of patient's survival. Overexpression of PTP4A3 in UM OCM-1 increased both cell migration and invasiveness.

We then got interested in identifying downstream targets of PTP4A3 and more precisely proteins linked to cytoskeleton. We first highlighted collapsin Response Mediator Protein (CRMP2) by 2-D electrophoresis of phosphorus-enriched UM OCM1 stably-expressing PTP4A3 or an inactive catalytic mutant of PTP4A3 (C104S). CRMP2 was dephosphorylated in presence of PTP4A3. CRMP2 expression in UM is inversely correlated with that of PTP4A3, suggesting an antagonistic role of these proteins in tumor progression. Through the use of shRNA in OCM-1, the downregulation of CRMP2 led to an increase in cell migration of PTP4A3-expressing OCM1. In addition, we focused on ATIP3, a protein encoded by the gene MTUS1, which binds to microtubules. In breast cancer, MTUS1 expression was shown to be downregulated in highly proliferative breast carcinomas of poor clinical outcome. Similarly, our transcriptomic analysis showed that expressions of PTP4A3 and MTUS1 were anti-correlated, suggesting that the reduction of MTUS1 gene and ATIP3 expression in UM would be of bad prognosis. In our OCM1 cells, we demonstrated that ATIP3 overexpression blocks proliferation however effect of ATIP3 overexpression on cell migration still need to be elucidaded.In conclusion, CRMP2 and ATIP3 may play a role in the metastatic process of UM tumors expressing high levels of the phosphatase.

Citation Format: Geraldine Liot, Oceane Anezo, Cecile Laurent, Selma Maacha, Nathalie Planque, Simon Saule. Potential targets of PTP4A3 involved in uveal melanoma migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4063. doi:10.1158/1538-7445.AM2014-4063
Abstract 4069: Capn4 contributes to tumor growth and metastasis of hepatocellular carcinoma by activation of FAK-Src signaling pathways
Authors: Dai Zhi , Zhou Jian , Fan Jia
Published: May 2015

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumor tissue from HCC patients compared to healthy patient tissue. Overexpression of Capn4 in HCC cells enhanced tumor cell growth in vitro and increased invasiveness, tumorigenicity, and lung metastasis in vivo. Protein microarray analyses in HCC cells with altered Capn4 levels (through Capn4 silencing or overexpression) showed that expression of multiple proteins was regulated by Capn4 and that the activation of focal adhesion kinase (FAK) signaling pathways might play a key role in Capn4-enhanced tumor development. Immunoprecipitation and western blotting verified that Capn4 was physically associated with FAK and promoted hyperactivity of the FAK-Src signaling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src, and p130Cas. Knockdown of Capn4 expression suppressed the malignant behavior of HCC cells and inhibited the FAK-Src signaling pathway. Furthermore, Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2 (MMP2) through activation of this signaling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FAK, and overexpression of both Capn4 and phospho-FAK correlates with the poorest survival outcomes in HCC. These results suggest that Capn4 can contribute to HCC growth and metastasis via activation of the FAK-Src Signaling pathway and MMP2. Capn4 could be a therapeutic target for HCC.

Citation Format: Zhi Dai, Jian Zhou, Jia Fan. Capn4 contributes to tumor growth and metastasis of hepatocellular carcinoma by activation of FAK-Src signaling pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4069. doi:10.1158/1538-7445.AM2014-4069
Abstract 4077: Impact of RET/PTC3 oncogene's inflammatory and transformative signaling on tumorigenicity of thyroid and non-thyroid tumorigenic cell lines
Authors: Kari Suresh C. , Eisenlohr Laurence C.
Published: May 2015

Rearranged in transformation/ papillary thyroid carcinoma 3 (RET/PTC3 or RP3), is a unique fusion oncogene, responsible for a benign version of PTC. RP3 has an active inflammatory component leading to the secretion of various pro-inflammatory cytokines, however the role of inflammation in development and progression of PTC has not been clearly defined. Facilitating investigation of this issue is our finding that the transforming and inflammatory signals of RP3 can be functionally separated allowing us to study each in isolation. We generated several RP3 mutants ablated in; 1) transformative signaling due to mutation of a tyrosine residue (RP3Y588Fmutant) which prevents the binding of downstream adaptor proteins 2) inflammatory signaling through mutation of binding sites for TRAF2 and TRAF6. Transformation only RP3 mutants grew tumors in immunodeficient and immunocompetent mice, strengthening our hypothesis that the inflammatory component of RP3 is responsible for the benign nature of PTC.

To assess the impact of inflammatory RP3 on the progression of different tumors, various thyroid and non-thyroid tumorigenic cell lines were transduced with MSCV_IRES_GFP_RP3 mutants and sorted for GFP expression. With highly immunogenic MC57G cells (C57BL/6 mice), the transforming only mutants developed tumors whereas wild type RP3 along with inflammatory only RP3 were rejected. Inflammation only RP3 mutant also led to slower tumor growth in Renca cell line (Balb/c) demonstrating the effect to be independent of haplotype. We are currently extending the analysis to PTC lines with BRAF and KRAS mutations, which are usually associated with more aggressive tumor profiles without an inflammatory tumor microenvironment. Our data so far suggest that contrary to the contemporary view, inflammation can retard tumor progression. Thus, the RP3 oncogene might be exploited in this regard. Future experiments aim to further characterize the importance of RP3-driven inflammation in tumor growth through continued in vivo and in vitro studies.

Citation Format: Suresh C. Kari, Laurence C. Eisenlohr. Impact of RET/PTC3 oncogene's inflammatory and transformative signaling on tumorigenicity of thyroid and non-thyroid tumorigenic cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4077. doi:10.1158/1538-7445.AM2014-4077
Abstract 4081: Id4 (inhibitor of dna binding 4) crosstalk with pten-akt pathway
Authors: Komaragiri Shravan Kumar
Published: May 2015

Introduction: Inhibitor of DNA binding/differentiation protein 4 (Id4), acts as a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. Id4 shares the homology of HLH domain with other Ids and lack the basic DNA binding region. Previous studies from our lab had shown that unlike Id1, Id2 and Id3, Id4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promoting apoptosis. In this study we investigated whether Id4 acts as a tumor suppressor by regulating the AKT pathway.

Experimental procedures: Id4 protein expression was analyzed on human prostate adenocarcinoma samples by Immunohistochemistry. We also performed Immunohistochemistry to determine AKT, pAKT, PTEN protein expression in Id4 knockout mouse prostates. In addition we determined the expression levels of the above mentioned proteins in prostate cancer cell lines with or without Id4 (LNCaP, LNCaP-Id4, DU145, DU145+Id4, PC3).

Results: IHC demonstrated decreased Id4 protein expression in human prostate tissue samples whereas higher nuclear Id4 expression was found in normal human and mouse prostate tissues. IHC on Id4 knockout (Id4-/-) mouse prostates demonstrated an increase in pAKT expression, whereas AKT expression did not change significantly when compared to normal mouse prostate. PTEN expression levels decreased ignificantly in Id4 knockout mouse prostates. Similar pattern was observed in prostate cancer cell lines: DU145 cells lacking Id4 had low PTEN as compared to Id4 over-expressing DU145 cells. Id4 expression was also associated with PI3 kinase activity in LNCaP and PC3 cells. These results suggest Id4 regulates AKT-PI3K pathway by attenuating PTEN.

Conclusions: Id4 appears as a potential tumor suppressor gene that exerts its tumor suppressor activity by promoting PTEN expression. PTEN in turn regulates AKT phosphorylation that stabilizes p53, AR and NKX3.1 expression in the normal prostate.

Acknowledgement: The research was supported by NIH/NCI-RO1CA128914 and in part by NIH/NCRR/RCMI G12RR03062

Citation Format: Shravan Kumar Komaragiri. Id4 (inhibitor of dna binding 4) crosstalk with pten-akt pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2014-4081
Abstract 4087: Identification of microRNAs involved in shaping immune surveillance and growth properties of tumors
Authors: Seliger Barbara , Jasinski-Bergner Simon , Bukur Juergen , Schulz Kristin , Stehle Franziska
Published: May 2015

Ligands of the natural killer (NK) cells and CD8+ cytotoxic T lymphocyte (CTL) receptors, such as the inhibitory classical HLA-G antigen, are often deregulated upon viral infection and neoplastic transformation thereby allowing virus-infected or malignant-transformed cells to escape NK and T cell-mediated immune surveillance. The underlying molecular mechanisms controlling the aberrant HLA-G expression have been recently identified suggesting that cellular microRNAs (miRs) target the 3′UTR of HLA-G mRNA thereby shaping the anti-tumoral immune response. Functional analysis of these miRs demonstrated an inverse correlation of HLA-G and miR expression, which was directly associated with tumor grading, staging, disease progression and survival of renal cell carcinoma patients. Furthermore, these miRs enable tumors to evade NK and T cell cytotoxicity. Using 2DE-based proteome analysis in combination with MALDI-TOF mass spectrometry of HLA-G-specific miR transfectants and control cells lead to the identification of novel targets of these miRs including the 14-3-3-β protein (YWHAB). The miR-regulated YWHAB expression not only alters tumor cell proliferation, but also the sensitivity to apoptosis. Loss of HLA-G-specific miRs enhanced YWHAB expression, which is accompanied by an inhibition of pro-apoptotic gene expression leading to apoptosis resistance, enhanced proliferation, migration and tumor progression. Furthermore, YWHAB expression and lack of HLA-G-specific miR expression directly correlate with a worse clinical outcome of HLA-G+ tumors linking for the first time tumor immune escape mechanisms with the malignant phenotype. Thus, the HLA-G-regulating miRs not only affect immune surveillance, but also enhance tumorigenicity.

Citation Format: Barbara Seliger, Simon Jasinski-Bergner, Juergen Bukur, Kristin Schulz, Franziska Stehle. Identification of microRNAs involved in shaping immune surveillance and growth properties of tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4087. doi:10.1158/1538-7445.AM2014-4087
Abstract 4088: Methylselenic acid inhibits the soluble and cell-surface bound NKG2D-ligand ULBP2 in melanoma
Authors: Uhlenbrock Franziska K. , Jensen Michael Hagemann , Kehlet Stephanie , Andresen Lars , Skov Søren
Published: May 2015

The secretion of the soluble NKG2D-ligand ULBP2 is a marker for poor prognosis in several types of cancer. NKG2D-ligands are normally directed to the cell surface of stressed, inflamed or pre-cancer cells and serve as targets for immune effector cells expressing the NKG2D receptor. The soluble NKG2D-ligands are devastating due to their chronic activation and thereby desensitization of NKG2D expressing immune effector cells. In fact this leads to severely compromised immune activation governing cancer development.

The present study describes that methylselenic acid (MSA) is able to inhibit soluble ULBP2 in different primary melanoma cell lines. Moreover, MSA actively suppressed ULBP2 both from constitutively expressing tumors and after induction by HDAC-inhibitors. In the attempt to elucidate the mechanism of inhibition, real-time qPCR experiments were performed. MSA activated the promoter and the transcription of the ULBP2 gene at a level similar to HDAC-inhibitors. These findings strongly indicate that ULBP2 is post-transcriptional regulated.

Selective blocking of ULBP2 from melanomas or other cancers that produces soluble immune suppressive ULBP2 is thus a new treatment strategy that also has the potential to be used in combination with existing treatment regimens.

Citation Format: Franziska K. Uhlenbrock, Michael Hagemann Jensen, Stephanie Kehlet, Lars Andresen, Søren Skov. Methylselenic acid inhibits the soluble and cell-surface bound NKG2D-ligand ULBP2 in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4088. doi:10.1158/1538-7445.AM2014-4088
Abstract 409: Regulation of chromatin remodeling in leukemia by Ikzf1 and Casein Kinase II
Authors: Payne Jonathon L. , Casiano Carlos M. , Payne Kimberly J. , Sloane Justin , Dovat Elanora , Song Chunhua , Dovat Sinisa
Published: May 2015

Ikzf1 (Ikaros) encodes a zinc finger protein that binds DNA and regulates gene expression via chromatin remodeling. The loss of Ikaros activity due to genetic or functional inactivation results in leukemia with a poor prognosis. The goal of our project is to determine the mechanism by which Ikaros regulates chromatin remodeling in human leukemia. Previous studies showed that Ikaros function in leukemia is controlled through its direct phosphorylation by Casein Kinase II (CK2). Treatment of leukemia cells with CK2 inhibitors results in enhanced Ikaros activity, which leads to cessation of cell growth. We have studied the mechanism by which inhibition of CK2 regulates Ikaros-induced epigenetic changes in leukemia. The human Nalm6 pre-B cell leukemia was treated with CK2 inhibitor, and the epigenetic signature of the histone modifications H3K9ac and H3K9me3 were determined using chromatin immunoprecipitation coupled with next generation sequencing (ChIP-SEQ). H3K9ac histone modification is associated with positive regulation of gene expression, while H3K9me3 is associated with the formation of heterochromatin and repression. The enrichment of particular histone modifications was confirmed by quantitative chromatin immunoprecipitation (qChIP). The results demonstrated that the inhibition of CK2 activity in leukemia results in a marked alteration in the epigenetic signature of both H3K9ac and H3K9me3 compared to untreated cells. This is associated with altered Ikaros binding to its target genes following CK2 inhibition. Current bioinformatics analysis is directed toward establishing a link between epigenetic modifications and Ikaros binding in leukemia. These results suggest that CK2 and Ikaros regulate gene transcription via epigenetic modifications and chromatin remodeling in leukemia.

Citation Format: Jonathon L. Payne, Carlos M. Casiano, Kimberly J. Payne, Justin Sloane, Elanora Dovat, Chunhua Song, Sinisa Dovat. Regulation of chromatin remodeling in leukemia by Ikzf1 and Casein Kinase II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 409. doi:10.1158/1538-7445.AM2014-409
Abstract 4105: Prevalence of breast stem cells in high-risk benign breast lesions and association with breastfeeding
Authors: Atkinson Rachel L. , Symmans Fraser , Bevers Therese B. , Woodward Wendy A. , Brewster Abenaa M.
Published: May 2015

Background: Invasive breast cancers contain a subpopulation of cancer stem cells that are thought to maintain the heterogeneity and self-renewing potential of the tumor. In our previously published data, we demonstrated that stem cells in normal adjacent tissues were associated with triple negative breast cancer. The objective of this study is to determine the presence of stem cells in high risk breast lesions. We hypothesized that stem cells in high-risk lesions and normal adjacent breast tissue may represent a biomarker for increased breast cancer risk. Methods: We identified biopsies from 42 high-risk women seen in the Cancer Prevention Center at The University of Texas MD Anderson Cancer Center between 1988-2011. We collected information on breast cancer risk factors. The biopsies contained high-risk lesions including ductal carcinoma in situ (DCIS) n=21, lobular carcinoma in situ (LCIS) n=5, atypical ductal hyperplasia (ADH) n=7 and atypical lobular hyperplasia (ALH) n=6. The tissues were fluorescently labeled for stem cell markers CD44/CD49f/CD133, and were scored positive if more then 1% cells were positive for all markers using an automated fluorescent microscopy software. The presence of stem cell markers was compared in the normal adjacent tissues and high-risk lesions from a subset of 25 patients. Results: We identified stem cells in 46% of samples containing DCIS, 67% of LCIS, 63% of ALH, and 70% of ADH. There was a 79% concordance in stem cell positive status between the high-risk lesion and adjacent normal tissue. Women with positive stem cell high-risk lesions had no significant difference in age at first birth (24.4 years vs 26.6 years p=0.3), parity (1.7 vs 1.9, p=0.5), or body mass index (27.8 vs 31.8, p=0.07) compared to women without positive stem cell high-risk lesions. Women with positive stem cell high-risk lesions had shorter breastfeeding duration than women without (2.4 months vs 9.0 months p= 0.05). Conclusion: This is the first report, to our knowledge, that stem cells are present in some high-risk lesions and their associated normal breast tissue. Correlation with future development of invasive breast cancer could lay the foundation for future targeted breast cancer prevention strategies.

Citation Format: Rachel L. Atkinson, Fraser Symmans, Therese B. Bevers, Wendy A. Woodward, Abenaa M. Brewster. Prevalence of breast stem cells in high-risk benign breast lesions and association with breastfeeding. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2014-4105
Abstract 4108: Efficacy of ellagic acid and its major urolithin metabolites in inhibiting growth of prostate cancer cells
Authors: Poudel Anil , Vadhanam Manicka V. , Burlison Joseph
Published: May 2015

Pomegranate juice, in the last several years, has gained tremendous attention for its proven ability to slow down prostate cancer as evident by reduced prostate-specific antigen doubling time. However, a bioactive compound responsible for this action has not yet been confirmed and thus, limited its chemo-preventive/therapeutic potential. Pharmacokinetic studies of oral consumption of pomegranate juice in human have shown partial hydrolysis of ellagitannins, major component in the juice, into ellagic acid which is subsequently converted to various urolithin metabolites by the gut microbial digestion. Urolithins are shown to have extended plasma half-life and are detected in urine up to 48 after oral consumption of pomegranate juice. We investigated the potential of ellagic acid and major urolithin metabolites; urolithin A and urolithin B, that were synthesized in our laboratory, in inhibiting the growth and survival of androgen-independent prostate cancer cells, PC-3 and DU-145 cells in vitro. We found significantly high dose and time-dependent inhibition of growth and proliferation of both cell lines by all three compounds tested. Ellagic acid was found to be the most potent inhibitor as evident by significantly lower IC50 of 8.17 µM and 1.57µM respectively for DU-145 and PC-3 cells after 48 hours. Urolithin B was more efficient than Urolithin A against DU-145 cells (IC50 of 17.42µM vs. 73.78µM) while it was reversed in PC-3 cells (IC50 of 92.25 µM vs. 36.96 µM). Similarly, these compounds also induced substantial levels of dose-dependent apoptosis in either cell lines, however, with variable sensitivity. Western blot analysis of signaling proteins to elucidate the molecular mechanisms of observed effects correlated with altered levels of proteins that mediate cell-cycle regulation. P21, an inhibitor of CDK2-CyclinA/E complex that regulates protein elongation and cell-cycle progression from G1 to S phase was highly upregulated in compound-treated PC-3 cells and DU-145 cells, while cyclinD1 which regulates G1 to S phase transition in consort with CDK4 was significantly decreased in treated cells in comparison to untreated and vehicle-treated control. Studies are underway to understand the synergistic effects between these compounds, in vitro and in vivo. Taken together, our results indicates EA and its gut metabolites are significant anti-survival and anti-proliferation candidates against prostate cancer cells and hold a tremendous chemo-therapeutic and chemo-preventive potential, for its high efficacy and natural, low toxic source.

Citation Format: Anil Poudel, Manicka V. Vadhanam, Joseph Burlison. Efficacy of ellagic acid and its major urolithin metabolites in inhibiting growth of prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2014-4108
Abstract 4117: Utilizing RNA aptamers for biomarker discovery in a novel cell culture system for hepatocellular carcinoma
Authors: Naqvi Ibtehaj A. , White Rebekah R. , Moylan Cynthia A. , Diehl Anna Mae , Choi Steve S.
Published: May 2015

Introduction: Current serum biomarkers for hepatocellular carcinoma (HCC) lack the sensitivity and specificity to be effective screening tools. Aptamers are single-stranded RNA oligonucleotides that specifically bind to target proteins with high affinity and are generated by repeatedly screening complex RNA libraries for binding to protein targets. We have utilized this selection process to identify biomarkers from complex targets, such as secreted proteomes (secretomes). Our hypothesis is that aptamers that specifically bind the HCC secretome will identify serum protein biomarkers for HCC.

Methods: We mirrored the microenvironments of HCC and normal liver tissue utilizing two co-culture systems: 1) human HCC cells (Huh-7) with normal hepatic stellate cells (HSCs) to model HCC livers and 2) normal human hepatocyte cells (THLE-2) with HSCs to model normal livers. Co-cultures were grown to confluence then switched to serum free media for collection of secreted cancer proteins (HCC secretome, HS) and normal secretome (NS). A nuclease-resistant RNA pool was then alternately incubated with NS (discarded for negative selection) and HS (recovered and amplified for positive selection).

Results: Binding affinity for HS increased over 7 successive rounds, while binding affinity for NS decreased. The final RNA pool was cloned and sequenced yielding 3 candidate aptamers that were tested for binding to the secretomes and to human serum from patients with HCC and normal controls. In the samples tested we found that one candidate aptamer sequence (G-20) demonstrated increased binding to the sera of HCC pts (N=5) relative to normal sera (N=2) and to a non-binding control aptamer. The maximal percent fraction bound of the G-20 aptamer to cancer serum was 15% compared to only 7% in control. The maximal percent fraction bound of G-20 in normal serum was 8% compared to 6% in control.

Conclusions: This co-culture system may better recapitulate the in vivo environment. Aptamers that specifically bind our cancer secretome over the normal secretome may bind to proteins that are specifically secreted by HCC tumors in vivo. The G-20 aptamer selectively binds to HCC patient sera. Future goals of this work are to utilize affinity purification techniques to identify the target of our G-20 aptamer and to continue testing our G-20 aptamer in patient sera to further validate its ability to differentiate between cancer and normal disease states.

Citation Format: Ibtehaj A. Naqvi, Rebekah R. White, Cynthia A. Moylan, Anna Mae Diehl, Steve S. Choi. Utilizing RNA aptamers for biomarker discovery in a novel cell culture system for hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4117. doi:10.1158/1538-7445.AM2014-4117
Abstract 4122: Anti-cancer activity of rosemary extract in colon cancer cells: Involvement of Nrf2 and ERK pathways
Authors: Yan Miao , Li Gongbo , Patiwala Sakina M. , Householter Emily , Johnson Jeremy J.
Published: May 2015

Background: The Mediterranean herb rosemary has been used for culinary purposes and for their medicinal properties for millennia. Rosemary and its polyphenolic diterpenes (carnosol and carnosic acid) are known to possess anti-oxidant activity that may be beneficial for cancer control. Colorectal cancer is the second leading cause of death from cancer in the United States. Nutrition, particularly intake of vegetables and certain plant components, has been reported to have a major role in colon cancer risk reduction. In our current study, we investigated the anticancer effects of rosemary extract and its two main active ingredients carnosol and carnosic acid on colon cancer cell lines to understand the mechanism. Next, we evaluated the anti-cancer activity of rosemary extract in a nude mice model.

Methods: Two human colon cancer cell lines, HCT116 and SW480, were treated with rosemary extract, carnosol or carnosic acid. MTT, TUNEL and ELISA were used to evaluate cell viability and apoptosis. Western blot and immunofluorescence were used to detect proteins involved in apoptosis. We also evaluated the anti-cancer activity of rosemary extract in nude mouse inoculated with HCT116 cells.

Results: Rosemary extract, carnosol and carnosic acid, increased apoptosis, decreased viability in colon cancer cell lines. Rosemary extract, carnosol and carnosic acid significantly upregulated the expression of Nrf2 and ERK. Rosemary extract also effectively suppressed tumor growth in a xenograft tumor model without affecting body weight.

Conclusions: Rosemary extract inhibits colon cancer cell growth in a xenograft mouse model without observable toxicity. Furthermore, rosemary extract, carnosol and carnosic acid induce the expression of Nrf2 and ERK pathways in colon cancer cells, suggesting further research is needed to evaluate its potential as an anti-cancer and/or chemoprevention agent.

Citation Format: Miao Yan, Gongbo Li, Sakina M. Patiwala, Emily Householter, Jeremy J. Johnson. Anti-cancer activity of rosemary extract in colon cancer cells: Involvement of Nrf2 and ERK pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4122. doi:10.1158/1538-7445.AM2014-4122
Abstract 413: Validation of methylation biomarkers that distinguish normal colon mucosa from cancer patients from normal colon mucosa of patients without cancer
Authors: Cesaroni Matteo
Published: May 2015

We have attempted to validate differences in DNA methylation levels of 30 candidate genes reported to discriminate between normal colon mucosa of colon cancer patients and normal colon mucosa of individuals without cancer. We found that CpG sites in 16 of the 30 candidate genes show significant differences in mean methylation level in normal colon mucosa in an independent cohort of 24 cancer patients and 24 controls. A support vector machine trained on these data and data for an additional 66 CpGs set yielded an 18-gene signature, composed of 10 of the validated candidate genes plus 8 additional candidates. This model exhibited 96% sensitivity and 100% specificity in a 40-sample training set and classified all eight samples in the test set correctly. Moreover, we found a moderate-strong correlation (Pearson coefficients r=0.253-0.722) between methylation levels in colon mucosa and methylation levels in peripheral blood for seven of the 18 genes in the support vector model. These 7 CpGs, alone, classified 44 of the 48 patients in the validation set correctly and five CpGs selected from only two of the seven genes classified 41 of the 48 patients in the discovery set correctly. These results suggest that methylation biomarkers may be developed that will, at minimum, serve as useful objective and quantitative diagnostic complements to colonoscopy as a cancer screening tool. These data also suggest that it may be possible to monitor biomarker methylation levels in tissues collected much less invasively than by colonoscopy.

Citation Format: Matteo Cesaroni. Validation of methylation biomarkers that distinguish normal colon mucosa from cancer patients from normal colon mucosa of patients without cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 413. doi:10.1158/1538-7445.AM2014-413
Abstract 4137: Latent class model characterization of neighborhood SES
Authors: Palumbo Aimee , Michael Yvonne , Hyslop Terry
Published: May 2015

Background: Breast cancer is the most common cancer in women and a significant source of mortality. Neighborhood-level socioeconomic status (NSES) has been shown to play a key role in health; poorer health outcomes are observed in deprived neighborhoods even after controlling for individual level SES. Commonly used NSES indexes are difficult to interpret. The relative impact of different factors within the index cannot be evaluated. Latent class models, which use latent variables to create distinct classes, allow for characterization of NSES and estimation of the effects of specific neighborhood characteristics on cancer outcomes.

Methods: We used data from women diagnosed with breast cancer from a teaching hospital in Philadelphia, PA. Census information at the Zip Code Tabulation Area (ZCTA) level was used to obtain NSES variables. Latent class analysis and comparisons of model fit statistics were used to determine the optimal number of classes.

Results: Complete ZCTA-level data was available for 1,664 breast cancer patients. In this population, NSES was best represented by two separate latent variables, each with 2-classes (LC2). When the class variables were compared to a continuous NSES index (NSI), the correlation was 0.85. However, LC2 demonstrated stronger association with race, stage, disease subtype, tumor size, and histologic grade.

Conclusions: We classified neighborhoods based on correlated socioeconomic census-level variables. Latent variables identify specific characteristics associated with living in a neighborhood with low versus high advantage and disadvantage and thus may improve our understanding of critical breast cancer prognostic factors and the targeting of cancer control efforts. Further research will provide overall structural latent models that incorporate information on tumor biology, prognostic variables and survival outcomes to aid in the identification of vulnerable populations.

Table 1. Neighborhood latent class model (LC2) and SES Index (NSI)

Table 1.Neighborhood latent class model (LC2) and SES Index (NSI)LC2 *ModelNSI Model *Prognostic Factor Stage24.5511.61 Race366.44331.01 Subtype18.4315.48 Size22.532.91* Chi-square statistic for association of Prognostic Factor with LC2 class or with NSI quartile. Higher number indicates higher strength of association.

Citation Format: Aimee Palumbo, Yvonne Michael, Terry Hyslop. Latent class model characterization of neighborhood SES. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4137. doi:10.1158/1538-7445.AM2014-4137
Abstract 414: Aberrant DNA methylation and expression of mRNA in EGFR-mutant lung cancer cell line with long-term exposure to gefitinib
Authors: Terai Hideki , Soejima Kenzo , Naoki Katsuhiko , Yasuda Hiroyuki , Sato Takashi , Arai Daisuke , Ohgino Keiko , Ishioka Kota , Kuroda Aoi , Tani Tetsuo , Ohashi Ayano , Nishino Makoto , Miyawaki Masayoshi , Hamamoto Junko , Betsuyaku Tomoko
Published: May 2015

Many of the patients with non-small cell lung cancer (NSCLC) who initially responded well to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) will eventually relapse. The mechanisms of resistance to EGFR-TKIs is not yet fully clarified. The suppression of genes by DNA methylation is reported to be involved in the mechanism of tolerance to cytotoxic drugs. The purpose of this study is to identify epigenetically regulated genes and to clarify the contribution of epigenetic alteration to the acquired resistance to EGFR-TKI. We established gefitinib-resistant PC-9, which was originally gefitinib-sensitive lung cancer cell line, by serial long term exposure to gefitinib. We collected RNA and DNA from both gefitinib-sensitive and -resistant PC-9 cells and performed comprehensive analysis for DNA methylation and mRNA expression using infinium array and cDNA microarray, respectively. We identified 640 genes those DNA methylations were increased in gefitinib-resistant cell line compared to parental cell line. Then, we selected 29 candidate genes those mRNA expression were decreased in resistant PC-9. We are currently underway to elucidate the specific function of each gene and updated data will be presented at the meeting.

Citation Format: Hideki Terai, Kenzo Soejima, Katsuhiko Naoki, Hiroyuki Yasuda, Takashi Sato, Daisuke Arai, Keiko Ohgino, Kota Ishioka, Aoi Kuroda, Tetsuo Tani, Ayano Ohashi, Makoto Nishino, Masayoshi Miyawaki, Junko Hamamoto, Tomoko Betsuyaku. Aberrant DNA methylation and expression of mRNA in EGFR-mutant lung cancer cell line with long-term exposure to gefitinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 414. doi:10.1158/1538-7445.AM2014-414