bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2081: FoxM1 transactivates PTTG1 and promotes colorectal cancer cell migration and invasion
Authors: Guo Jinjun , Zheng Yun , Tong Yunguang , Koeffler H Philip
Published: May 2015

Previous studies have shown that both Forkhead Box M1 (FoxM1) and pituitary tumor transforming gene 1 (PTTG1) were associated with cancer development and metastases. This study aims to investigate the relation between FoxM1 and PTTG1 and its role in colorectal cancer cell behavior.

The microarray data of colorectal cancer were analyzed to determine the association of FoxM1 and PTTG1 expression. Luciferase reporter assay, ChIP and EMSA were used to identify the regulatory mechanisms between FoxM1 and PTTG1. PTTG1 mRNA and protein levels were examined using real-time PCR and western blot after knocking down or over expressing FoxM1. The roles of FoxM1 and PTTG in tumor metastasis were investigated by cell migration and invasion assays.

Gene expression microarray data indicated that FoxM1 and PTTG1 are concordantly up-regulated in colorectal cancer tissues. Over-expression of FoxM1 up-regulated PTTG1 expression and knock-down of FoxM1 did the opposite, indicating that PTTG1 was regulated by FoxM1. Luciferase reporter assay indicated that -391 to +50 bps of PTTG1 promoter are important for FoxM1 to enhance PTTG1 promoter activity. Sequence analysis identified two potential FoxM1 binding sites. EMSA indicated that FoxM1 directly binds to PTTG1 promoter at the -350 to -343 binding motif. This binding was confirmed by chromatin immunoprecipitation in HCT116 and SW620 cells. Boyden chamber assay indicated that FoxM1 and PTTG1 concordantly regulate migration and invasion of HCT116 and SW620 colorectal cancer cells.

In summary, our results elucidate that FoxM1 up-regulates PTTG1 transcription through binding to PTTG1 promoter. PTTG1 likely mediates effects of FoxM1 on colorectal cancer cell migration and invasion.

Citation Format: Jinjun Guo, Yun Zheng, Yunguang Tong, H Philip Koeffler. FoxM1 transactivates PTTG1 and promotes colorectal cancer cell migration and invasion. [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 2081. doi:10.1158/1538-7445.AM2014-2081
Abstract 2082: PHD3 regulates EMT and therapy response in lung cancer
Authors: Gonzalez Jose Higinio Dopeso
Published: May 2015

The hypoxic microenvironment is a characteristic feature of solid tumors. Hypoxia initiates a number of adaptive responses that collectively promote a more aggressive tumor phenotype. One important process that is activated by hypoxia is epithelial-mesenchymal transition (EMT), a cellular program that induces cell detachment and increases the invasive capacity of the cells. The prolyl hydroxylase domain proteins PHD1, 2 and 3 play a central role in adaption to hypoxia and act as oxygen sensors that regulate the hypoxia-inducible factors (HIFs) response. Here, we identify PHD3 as a crucial regulator of central tumor hallmarks in lung cancer. We show that PHD3 is regulated during EMT and that modulation of PHD3 levels in lung carcinoma cells controls EMT, the tumorigenicity and metastatic capacity of lung cancer cells in various animal tumor models. Mechanistically we show that these functions of PHD3 are mediated through a signaling cascade leading to increased SMAD activity. Moreover, we show that these effects are mechanistically linked to the acquisition of therapeutic resistance in lung cancer and may therefore serve to predict therapy response. These findings highlight PHD3 as an important regulator of lung cancer progression, which controls several cancer hallmarks including growth, invasion, metastasis and therapy resistance.

Citation Format: Jose Higinio Dopeso Gonzalez. PHD3 regulates EMT and therapy response in lung 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 2082. doi:10.1158/1538-7445.AM2014-2082
Abstract 2090: Novel role of STAT-3 in anoikis resistance and metastasis in melanoma cells in vitro and in vivo
Authors: Fofaria Neel Manoj , Srivastava Sanjay K.
Published: May 2015

Anoikis is an anchorage-independent cell death. Resistance to anoikis is one of the key features of metastatic cells. The role of STAT-3 in anoikis resistance in various melanoma cells was analyzed in the current study. Significant proportion of SK-MEL-28, SK-MEL-2, MeWo, SK-MEL-5 and B16-F0 cells resisted anoikis when grown under anchorage-independent conditions. Cells that resisted anoikis were observed to a have higher rate of migration and invasion as compared to the cells grown under anchorage dependent conditions. The anoikis resistant cells also exhibited significantly higher expression and phosphorylation of STAT-3 at Tyr 705 than the cells that were attached to the basement membrane. Treatment of these cells with IL-6, a cytokine which phosphorylates STAT-3, prevented the induction of anoikis. STAT-3 inhibitors AG490 and PL induced anoikis in a concentration-dependent manner in anoikis resistant cells, whereas IL-6 blocked anoikis. Over-expression of STAT-3 by transfection in SK-MEL-28 and SK-MEL-2 cells significantly increased the anoikis resistance, confirming the role of STAT-3 in anoikis resistance. On the other hand, silencing STAT-3 in SK-MEL-28, SK-MEL-2, SK-MEL-5 and MeWo cells decreased the potential of cancer cells to resist anoikis. Furthermore, STAT-3 (-/-) melanoma cells were more sensitive to anoikis, as compared to cells overexpressing STAT-3 (+/+) cells. STAT-3 (-/-) SK-MEL-28 cells and the SK-MEL-28 cells that were treated with PL completely failed to develop tumors when injected subcutaneously in immune-compromised mice. Moreover, these cells also failed to metastasize when injected intravenously. On the other hand, anoikis resistant wild type cells not only formed aggressive tumors subcutaneously but also highly metastasized when injected intravenously. The metastasis was confirmed by H & E staining. In summary, our results establish STAT-3 as a critical player that renders anoikis resistance to pancreatic cancer cells and enhance their metastasis potential. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: Neel Manoj Fofaria, Sanjay K. Srivastava. Novel role of STAT-3 in anoikis resistance and metastasis in melanoma cells in vitro and in vivo. [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 2090. doi:10.1158/1538-7445.AM2014-2090
Abstract 21: Stromal fibroblasts in breast carcinomas promote angiogenisis through Adrenomedullin (AM) secretion
Authors: Benyahia Zohra
Published: May 2015

Tumor stroma is an active part influencing the biological properties of malignancies via molecular cross-talk. Tumor- or cancer-associated fibroblasts (TAFs or CAFs) play a significant role in this interaction. These cells frequently express smooth muscle actin and can be classified as myofibroblasts. CAFs often constitute the majority of the stromal cells within a breast carcinoma. Adrenomedullin (AM), a multifunctional peptide, is highly expressed in several tumors and plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). In this study, RT-QPCR showed the expression of AM and AM receptors mRNAs in CAFs and breast carcinoma cell lines (MCF7 and MDA 231). Immunostaining demonstrates the presence of immunoreactive AM in all the CAFs analyzed (n=15). AM activates MAP kinase pathway by inducing a phosphorylation of ERK1/2 and p38 in both cell types suggesting a cross-talk between tumor cells and CAFs ensured by AM. In vivo, CAFs in Matrigel plugs induce angiogenesis by promoting recruitment of endothelial cells, pericytes, myeloid precursor cells and by promoting channel formation. AM and AM receptors neutralizing antibodies blocked neovascularisation of Matrigel plugs suggesting strongly that angiogenesis induced by CAFs into carcinoma are mediated in part by AM. Our findings indicate that CAFs within breast carcinomas may contribute to tumor promotion in part through the secretion of AM.

Note: This abstract was not presented at the meeting.

Citation Format: Zohra Benyahia. Stromal fibroblasts in breast carcinomas promote angiogenisis through Adrenomedullin (AM) secretion. [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 21. doi:10.1158/1538-7445.AM2014-21
Abstract 210: Id4 inhibition of twist1-mediated autocrine PDGF signaling inhibits growth and promotes apoptosis of glioblastoma-derived stem cell cultures
Authors: Rahme Gilbert J. , Israel Mark A.
Published: May 2015

Glioblastoma Multiforme (GBM) is the most common, aggressive, and lethal primary brain tumor. A hallmark of GBM is the resistance of tumor stem cells to chemotherapy resulting in tumor recurrence and progression decreasing the quality of life and causing patient death. The prevention of GBM recurrence after chemotherapy by eliminating cancer stem cells enhances survival in genetically engineered mouse models; however, the signal transduction networks that control cancer stem cell survival and proliferation remain poorly understood.

We have discovered that decreased expression of Id4, a dominant negative helix-loop-helix transcription factor, is associated with the proliferation and survival of sphere cultures from GBM-derived cell lines, which are enriched for cancer stem cells. In striking contrast, Id4 expression did not affect proliferation or apoptosis in differentiated, adherent cultures of these same cell lines. Moreover, the cell lines we examined required autocrine PDGF signaling for growth. We found that Id4 inhibited autocrine PDGF signaling in cancer stem cell enriched cultures by suppressing the expression of platelet-derived growth factor receptor α (PDGFRα). In these studies we found that Id4 mediated inhibition of PDGFRα expression was mediated through the antagonistic interaction between Id4 and Twist1, a basic helix-loop-helix transcription factor. This observation is consistent with our previously reported finding that Id4, uniquely among the Id family of proteins, binds to and antagonizes Twist1. Further, using the Rembrandt and TCGA databases, we found that Id4 expression levels directly correlate with glioma patient survival and can predict the level of PDGFRα expression. These data provide strong evidence that Id4 may regulate GBM stem cell survival and thereby contribute to GBM tumor progression. Along with our recent report demonstrating that Id4 can block invasion of GBM by silencing Twist1-mediated MMP2 expression, these findings suggest that pharmacological targeting of the Id4/Twist1 complex might provide opportunities to reduce the lethality of GBM by regulating both GBM invasion and tumor stem cell maintenance.

Citation Format: Gilbert J. Rahme, Mark A. Israel. Id4 inhibition of twist1-mediated autocrine PDGF signaling inhibits growth and promotes apoptosis of glioblastoma-derived stem cell cultures. [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 210. doi:10.1158/1538-7445.AM2014-210
Abstract 2101: Role of CDK8 in estrogen receptor signaling in breast cancers
Authors: McDermott Martina , Gyorffy Balazs , Lim Chang-uk , Chumanevich Alexander , Yang Zhengguan , Chen Mengqian , Catroppo James F. , Roninson Igor , Broude Eugenia V.
Published: May 2015

The majority of breast cancers (BrCa) overexpress estrogen receptor (ER)α, which regulates transcription and drives estrogen-stimulated proliferation of ER+ tumor cells. ER+ patients usually receive adjuvant anti-estrogen therapy based on ER modification, downregulation, or estrogen depletion. Tumors frequently develop resistance to anti-estrogen therapy through various mechanisms, which may involve stimulation of ER itself or of downstream mediators of ER-driven transcription, as well as activation of alternative proliferation pathways, in particular those driven by HER2/EGFR. The treatment efficacy of hormone-resistant BrCa could be greatly augmented by targeting new druggable mediators of ER activity. We have now identified a transcription-regulating oncogenic kinase CDK8 as a potentiator of ER signaling and ER-driven BrCa cell proliferation. Immunohistochemical staining of breast tissue arrays and bioinformatics analysis of gene expression microarray data of breast cancer patients revealed that CDK8 is overexpressed in BrCa and that higher CDK8 expression correlates with the failure of systemic therapy. Among systemically untreated patients, higher CDK8 expression was correlated with shorter relapse-free survival in a subset of ER+ tumors that expressed the lowest levels of ERα. This correlation suggested that CDK8 could play a role in the progression of ER+ breast cancers with reduced levels of ER, possibly by stimulating the mitogenic effects of ER-mediated transcription. Indeed, a selective small-molecule CDK8 inhibitor (Senexin A) decreased estrogen-induced ER-dependent transcription and inhibited estrogen-stimulated proliferation of ER+ BrCa cell lines. Microarray analysis revealed that CDK8 inhibition diminished the induction of genes that show rapid and sustained activation by estrogen in ER+ cells. CDK8 inhibition had an additive effect in combination with anti-estrogens in ER+ BrCa and a synergistic effect with fulvestrant in BrCa cells resistant to estrogen deprivation. Some of these cell lines also express HER2/EGFR, and CDK8 inhibition in combination with a HER2/EGFR inhibitor lapatinib synergistically inhibited the growth of these cells. These results suggest that combining anti-estrogen and anti-CDK8 therapy may be more effective than conventional hormone therapy for ER+ BrCa.

Citation Format: Martina McDermott, Balazs Gyorffy, Chang-uk Lim, Alexander Chumanevich, Zhengguan Yang, Mengqian Chen, James F. Catroppo, Igor Roninson, Eugenia V. Broude. Role of CDK8 in estrogen receptor signaling in breast cancers. [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 2101. doi:10.1158/1538-7445.AM2014-2101
Abstract 2106: Estrogen receptor alpha drives proliferation of prostate cancer through PI3K and MAPK signaling
Authors: Takizawa Itsuhiro , Lawrence Mitchell , Pearson Helen , Pedersen John , Pouliot Normand ,   Australian Prostate Cancer BioResource, Humbert Patrick , Furic Luc , Risbridger Gail
Published: May 2015

Prostate cancer, a hormone-dependent disease of aging men, occurs as the relative levels of estrogen compared to testosterone are increasing. Indeed, high doses of estrogen, in combination with androgens, can initiate prostate cancer. This effect is attributed to the activity of the estrogen receptor α (ERα), but a paucity of suitable models means that the precise role of ERα in prostate cancer is still poorly understood.

In this study we observed increased ERα expression in three different models of prostate cancer, PTEN null mice, Hi-Myc mice, and high grade human tumor specimens. Within the PTEN null prostate, there was a consistent pattern of ERα expression: low in benign glands, moderate in tumors within the dorsal, lateral and ventral lobes, and high in tumors within the anterior prostate. This pattern significantly correlated with the levels of the proliferative marker Ki67. There was also a significant correlation between ERα and Ki67 within individual malignant glands in the anterior prostate. Furthermore, we demonstrated that ERα sustained the in vitro proliferation of cells derived from a PTEN null tumor in 2D and 3D assays. There was a significant decrease in proliferation in cells treated with TPSF, a non-competitive ERα antagonist, or with ERα-specific shRNA. Loss of ERα caused a significant decrease in the levels of MYC and other ERα target genes. It also reduced the activity of both the PI3K and MAPK pathways as measured by decreased levels of phosphorylated erk1/2, S6 kinase and other downstream factors. This effect was reversed in rescue experiments with expression constructs for both full length ERα, capable of genomic and non-genomic actions, and membrane-only ERα, only able to trigger rapid non-genomic signalling.

Collectively, these results demonstrate that ERα levels increase in prostate cancer, which promotes proliferation through classical genomic and rapid non-genomic signalling.

Citation Format: Itsuhiro Takizawa, Mitchell Lawrence, Helen Pearson, John Pedersen, Normand Pouliot, Australian Prostate Cancer BioResource, Patrick Humbert, Luc Furic, Gail Risbridger. Estrogen receptor alpha drives proliferation of prostate cancer through PI3K and MAPK signaling. [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 2106. doi:10.1158/1538-7445.AM2014-2106
Abstract 2108: Estrogen non genomic signalling is activated in tamoxifen-resistant breast cancer
Authors: Poulard Coralie , Rambaud Juliette , Cohen Pascale , Migliaccio Antimo , Corbo Laura , Marangoni Elisabetta , Romancer Muriel L.
Published: May 2015

Introduction: Although the presence of nuclear estrogen receptor (ERα) is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signalling. We have already shown that arginine methylation of ERα is essential for E2-non genomic pathway triggered by ERα interaction with PI3K and c-Src thereby propagating the signal to downstream transduction cascades (Le Romancer et al, Mol Cell, 2008). Recently, we have found that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy (Poulard et al, Embo Mol Med, 2012). In addition, the cross talk between ERα and growth factor signalling has emerged as a critical factor in endocrine resistance. The aim of this study was to investigate the receptor cytoplasmic signalling in human ERα-positive breast cancer with acquired resistance to tamoxifen.

Experimental procedures: The activation of cytoplasmic ER signalling pathway was investigated in tamoxifen resistant MCF-7 cells and in patient-derived xenografts (PDX) of ERα-positive breast cancer with acquired resistance in vivo to tamoxifen. In addition, tamoxifen-resistant PDX models were treated with tamoxifen, fulvestrant and everolimus, during 90 days. ERα interactions with Src and PI3K were investigated by Proximity Ligation Assay (PLA) in formalin-fixed tumors, a technology which enables to investigate protein/protein interactions in tissues.

Results: Interestingly, we detected an increase of ERα/Src/PI3K expression in tamoxifen resistant versus parental MCF-7 cells. This result was confirmed in one PDX model where we found a significant increase of ERα/Src/PI3K expression in the tamoxifen-resistant tumor compared to the corresponding parental xenograft. Finally, the analysis of treated tumors revealed a significant decrease of ERα/Src/PI3K expression in the fulvestrant-treated tumors as well as in tumors treated by the everolimus and fulvestrant combination.

In conclusion these results show that ERα cytoplasmic signalling is associated to acquisition of tamoxifen resistance both in cell line and patients-derived xenografts. In addition, ERα cytoplasmic signalling appears to be affected in tumors responding to alternative treatments such as fulvestrant or everolimus. Targeted therapies against ERα, Src and PI3K are in progress and could constitute a new approach to circumvent resistance to endocrine therapies.

Citation Format: Coralie Poulard, Juliette Rambaud, Pascale Cohen, Antimo Migliaccio, Laura Corbo, Elisabetta Marangoni, Muriel Le Romancer. Estrogen non genomic signalling is activated in tamoxifen-resistant 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 2108. doi:10.1158/1538-7445.AM2014-2108
Abstract 2110: Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7
Authors: Kimura Mariko , Hanamura Toru , Niwa Toshifumi , Yamaguchi Yuri , Endo Itaru , Hayashi Shin-ichi
Published: May 2015

mTOR inhibitor everolimus has been considered as a promising agent for breast cancer treatment especially among estrogen receptor (ER) positive postmenopausal advanced breast cancer. As expected, combination therapy of everolimus and steroidal aromatase inhibitor (AI) showed remarkable clinical efficacy for the first line nonsteroidal AI treatment failure patients in BOLERO-2 trial. However, the subsequent treatment after refractory to that therapy remains a great matter. We already reported several types of long term estrogen-depletion resistant (EDR) cell lines from MCF-7-E10, which has been constitutively ERE-GFP-transfected to monitor ER activity in living cells (Fujiki N et al., J Steroid Biochem Mol Biol. 2013 Oct 13;139C:61-72). They mimic AI resistance and these EDR cells have been characterized as the followings. 1) Type 1 as ER positive (ERE-GFP positive), constitutive ER overexpression without estrogen, and PI3K/Akt/mTOR pathway upregulated. 2) Type 2 as ER negative conversion (ERE-GFP negative) and IGF-1R/JNK signaling pathway upregulated. Then with those two types of sub-cell lines, we generated everolimus resistant cells after long term exposure to everolimus in estrogen depleted medium. Those everolimus resistant (EVR) cells are equivalent to resistance to the combination therapy of AI and everolimus as the second line endocrine treatment after the first line AI treatment failure. Though everolimus extremely inhibited cell proliferation of two EDR cell lines, everolimus resistant cells named as Type 1-EVR, Type 2-EVR no longer responded to everolimus. ER expression and estrogen sensitivity of two types of EVR cells were similar to those of the parental cells, respectively. Pure anti-estrogen fulvestrant inhibited cell proliferation of Type1-EVR cells. On the other hand, Type2-EVR did not respond to fulvestrant but JNK inhibitor inhibited the cell growth. Phosphorylation status of p70S6K and 4E-BP1 were different among individual parental EDR cells and EVR cells. Type1-EVR showed much higher expression of phosphorylation of MAP-kinase than the parental cells. In contrast, Type2-EVR expressed phosphorylation of MAP-kinase less than the parental cells. As above, the mechanism of acquired resistance to everolimus was likely to be different between those two sub-cell lines. This finding implies that different mechanisms of AI resistance follow different types of everolimus resistance.

Citation Format: Mariko Kimura, Toru Hanamura, Toshifumi Niwa, Yuri Yamaguchi, Itaru Endo, Shin-ichi Hayashi. Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7. [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 2110. doi:10.1158/1538-7445.AM2014-2110
Abstract 2111: Role of gene repression by estrogen in invasiveness of breast cancer cells
Authors: Patki Mugdha , Salazar Marcela d'Alincourt , Trumbly Robert , Ratnam Manohar
Published: May 2015

More than 75% of breast tumors are estrogen receptor (ER)-positive and require estrogen for their growth and survival. Long-term adjuvant therapies that disrupt estrogen signaling, in particular tamoxifen and aromatase inhibitors, are a mainstay in breast cancer treatment. The principal focus of mechanistic studies evaluating the role of estrogen in breast cancer proliferation has been on gene activation by estrogen and its antagonism by anti-estrogens. Gene repression by estrogen and its antagonism by anti-estrogens have unclear physiological and therapeutic consequence in breast cancer. In this study, we found that estrogen-repressed genes but not estrogen-activated genes overlapped the gene overexpression signature of clinical progression of ductal carcinoma in situ to invasive ductal carcinoma. An extensive gene ontology analysis revealed that genes repressed by estrogen are enriched for functions known to collectively support breast cancer progression. In ER-positive breast cancer cells tamoxifen prevented gene repression by estrogen. In these cells, estrogen inhibited invasiveness in a manner that was opposed by tamoxifen. These effects of estrogen and tamoxifen on gene repression and invasiveness also occurred in Her2+/ER+ cells whose growth was hormone-independent and insensitive to tamoxifen. The effects were also independent of ErbB2 gene regulation by estrogen. Several ER co-repressors supported repression of the tumor progression genes by estrogen. Gene repression by estrogen was causally linked to inhibition of invasiveness as a co-repressor binding site mutation in ER that selectively disrupted the ability of estrogen to repress genes also prevented inhibition of invasiveness. Gene repression by estrogen involved ER binding at non-classical chromatin sites that was prevented by tamoxifen. The findings offer a mechanistic understanding of the invasive potential of ER+ breast cancer cells that have evaded growth inhibition during conventional anti-estrogen treatments.

Citation Format: Mugdha Patki, Marcela d'Alincourt Salazar, Robert Trumbly, Manohar Ratnam. Role of gene repression by estrogen in invasiveness of breast 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 2111. doi:10.1158/1538-7445.AM2014-2111
Abstract 2113: Androgen receptor splice variants activating the full-length receptor in mediating resistance to androgen-directed therapy
Authors: Qi Yanfeng , Cao Bo , Zhang Guanyi , Xu Duo , Guo Zhiyong , Xiong Zhenggang , Plymate Stephen , Sartor Oliver , Zhang Haitao , Dong Yan
Published: May 2015

Androgen deprivation therapy, which disrupts androgen receptor (AR) signaling through androgen ablation or AR antagonists, is the first-line treatment for disseminated prostate cancer. While this regimen is effective initially, progression to the presently incurable and lethal stage, termed castration-resistant prostate cancer, invariably occurs. Upregulation of constitutively-active AR splice variants (AR-Vs) has been implicated in AR-driven tumor progression in castration-resistant prostate cancer. To date, functional studies of AR-Vs have been focused mainly on their ability to regulate gene expression independent of the full-length AR (AR-FL). Here, we showed that AR-V7 and ARv567es, two major AR-Vs, both facilitated AR-FL nuclear translocation in the absence of androgen and mitigated the ability of the antiandrogen enzalutamide to inhibit AR-FL nuclear trafficking. AR-V bound to the promoter of its specific target without AR-FL, but when dimerized with AR-FL, co-occupied the promoter of canonical AR target with AR-FL in a mutually-dependent manner. AR-V expression attenuated both androgen and enzalutamide modulation of AR-FL activity/cell growth, and mitigated the in vivo antitumor efficacy of enzalutamide. Furthermore, AR-V levels were upregulated in xenograft tumors that had acquired enzalutamide resistance. Collectively, this study highlights a dual function of AR-Vs in mediating castration resistance. In addition to trans-activating target genes independent of AR-FL, AR-Vs can serve as a “rheostat” to control the response of AR-FL to androgen-directed therapy via activating AR-FL in an androgen-independent manner. The findings shed new insights into the mechanisms of AR-V-mediated castration resistance and have significant therapeutic implications.

Citation Format: Yanfeng Qi, Bo Cao, Guanyi Zhang, Duo Xu, Zhiyong Guo, Zhenggang Xiong, Stephen Plymate, Oliver Sartor, Haitao Zhang, Yan Dong. Androgen receptor splice variants activating the full-length receptor in mediating resistance to androgen-directed therapy. [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 2113. doi:10.1158/1538-7445.AM2014-2113
Abstract 2117: Constitutively active androgen receptor variants upregulate expression of mesenchymal markers in prostate cancer cells
Authors: COTTARD Félicie , ASMANE Irène , ERDMANN Eva , BERGERAT Jean-Pierre , KURTZ Jean-Emmanuel , CERALINE Jocelyn
Published: May 2015

Androgen receptor (AR) signaling pathway remains the main target of novel therapeutics for castration-resistant prostate cancer (CRPC). However, constitutively active AR variants lacking the carboxy-terminal region in CRPC lead to therapy inefficacy. Moreover, recent studies suggest that AR variants are expressed in primary prostate tumors and may contribute to tumor progression. The aim of this study was to investigate the impact of AR variants on tumor progression.

N-cadherin expression was analyzed in LNCaP cells overexpressing the wild type AR or a constitutively active AR variant by qRT-PCR, Western blot and immunofluorescence. We showed here for the first time that N-cadherin expression was increased in the presence of constitutively active AR variants. Moreover, this increased expression of N-cadherin in LNCaP overexpressing AR variants was negatively regulated by androgens.

Although N-cadherin expression is often associated with a downregulation of E-cadherin, this phenomenon was not observed in our model. Nevertheless, in addition to the increased expression of N-cadherin, an upregulation of other mesenchymal markers expression such as Vimentin, SNAIL and ZEB1 was observed in the presence of constitutively active AR variants.

In conclusion, our findings highlight novel consequences of constitutively active AR variants on the regulation of mesenchymal markers in prostate cancer.

Citation Format: Félicie COTTARD, Irène ASMANE, Eva ERDMANN, Jean-Pierre BERGERAT, Jean-Emmanuel KURTZ, Jocelyn CERALINE. Constitutively active androgen receptor variants upregulate expression of mesenchymal markers in 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 2117. doi:10.1158/1538-7445.AM2014-2117
Abstract 2119: Regulation of T-type calcium channel expression during IL-6 induced neuroendocrine differentiation of prostate cancer cells
Authors: Weaver Erika , Hearne Jennifer L. , Martin-Caraballo Miguel
Published: May 2015

Prostate cancer is the most common cancer among men. At early stages, the growth of prostate cancers is androgen dependent and therefore therapies designed to reduce the androgen concentration/receptor activation are effective in promoting tumor regression. At later stages, however, the growth of prostate cancers becomes androgen independent, leading to increased mortality. The switch to an androgen-refractory state is associated with neuroendocrine differentiation of prostate cancer cells. Several factors including interleukin-6 (IL-6) and increased cAMP production promote neuroendocrine differentiation of prostate cancer cells. How these factors promote neuroendocrine differentiation is not completely understood. In this work we investigated whether IL-6 evoked neuroendocrine differentiation of prostate cancer cells results in the stimulation of T-type calcium channel expression in the prostate cancer cell line LNCaP. Treatment of LNCaP cells with IL-6 for four days evokes considerable morphological and molecular changes consistent with neuroendocrine differentiation, including the presence of rounded cell bodies, the appearance of long dendritic-like processes and the expression of chromogranin-A. IL-6 evoked neuroendocrine differentiation of LNCaP cells results in a 3-fold increase in the protein expression of the T-type calcium channel subunit Cav3.2. Transcripts for the Cav3.2 but not Cav3.1 or Cav3.3 subunits can be detected in IL-6-treated LNCaP cells. Real time PCR analysis indicates no change in Cav3.2 mRNA expression between control (non-stimulated) and IL-6 stimulated LNCaP cells, suggesting that T-type calcium channel expression is regulated by a post-transcriptional mechanism. Electrophysiological recordings reveal that increased Cav3.2 protein expression following IL-6 stimulation of LNCaP cells does not result in increased expression of functional channels in the membrane. Functional expression of Cav3.2 channels is facilitated by stimulation of LNCaP cells with forskolin an agent that increases intracellular cAMP. These results indicate that changes in T-type calcium channel expression and intracellular calcium during neuroendocrine differentiation of LNCaP cells are regulated by the interplay of multiple factors. Thus, it appears that T-type calcium channels could be a target for future therapeutic strategies against prostate cancers refractory to anti-androgen therapies.

Citation Format: Erika Weaver, Jennifer L. Hearne, Miguel Martin-Caraballo. Regulation of T-type calcium channel expression during IL-6 induced neuroendocrine differentiation 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 2119. doi:10.1158/1538-7445.AM2014-2119
Abstract 2121: Resveratrol regulates phospho-serine 81 androgen receptor and its stability to inhibit growth of prostate cancer cells
Authors: Peng Yu-Ting , Chen Tzu-Yin , Chen Mei-Chih , Lin Eugene , Lin Ho
Published: May 2015

Resveratrol, a polyphenol, has been reported as a cell growth inhibitor of various types of cancer. The molecular mechanisms of resveratrol are involved in different cellular signal transductions such as anti-inflammation and anti-oxidation. Resveratrol inhibits growth of prostate cancer cells through regulating cyclin-dependent kinases, anti-apoptotic proteins, and pro-apoptotic proteins. Notably, resveratrol may down-regulate androgen receptor (AR) and its downstream gene to inhibit prostate cancer cell growth. In previous study, serine 81 phosphorylation of AR increases its protein stability and transcriptional activity, which promotes prostate cancer cell growth. The aim of this study is to investigate whether AR serine 81 phosphorylation would be a target of resveratrol in prostate cancer cells. Our data indicate that resveratrol inhibited the growth of prostate cancer cell line, LNCaP, with the decrease of AR phospho-serine 81 levels. Moreover, AR protein stability was declined by resveratrol without affecting AR mRNA expression. Indeed, resveratrol inhibited AR transcriptional activity in results of AR reporter assay and PSA level in LNCaP cells. In summary, the results suggest that resveratrol might inhibit growth of LNCaP cells, at least, via decreasing phospho-serine 81 of AR, AR protein stability, and AR transcriptional activity.

Citation Format: Yu-Ting Peng, Tzu-Yin Chen, Mei-Chih Chen, Eugene Lin, Ho Lin. Resveratrol regulates phospho-serine 81 androgen receptor and its stability to inhibit 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 2121. doi:10.1158/1538-7445.AM2014-2121
Abstract 2122: Androgen decreases proliferation of thyroid cancer cells
Authors: MacEwan Melanie Elizabeth , O'Connell Timmy , Zhao Hong , Iacob Codrin , Suslina Nina , Moscatello Augustine , Shin Edward , Tiwari Raj , Darzynkiewicz Zbigniew , Geliebter Jan
Published: May 2015

The incidence of thyroid cancer in the United States has significantly increased over the past decade, with over 60,220 cases predicted for 2013. While premenopausal women have an approximately four fold higher incidence of papillary thyroid cancer (PTC) than men, once diagnosed, PTC in men exhibits increased aggressiveness and results in poorer clinical outcomes. These paradoxical observations led us to investigate the role that androgens and/or androgen receptors play in PTC. Analysis of AR expression in 24 PTC patient tissue samples indicated a 5.97 fold reduction in AR expression compared to matched, normal tissue (P<0.0001). To more fully investigate the function of androgen/AR in thyroid cells in a more controlled experimental system, the AR cDNA (pSG5-AR) was subcloned into the pcDNA3.1+ expression vector and stably transfected into 8505C anaplastic/PTC cells. Addition of DHT to the 8505C-transfected clone, 84E7, resulted in AR translocation into the nucleus and a 48% reduction in proliferation (p=0.01) at 72 hours, as well as a shift in the cell cycle toward G1 arrest during the same time period. Transcription profiling using RNA-Seq and gene ontology analysis revealed significant changes in the process of cell proliferation (p=2.47e-24) possibly due to significant changes in CDK1A/p21 (up 3.42 fold), RhoB (up 50 fold), Jun (down 1.7 fold), SCF (down 3.31 fold) and NFKBIA (up 2.36). Additionally, expression levels of other cell cycle proteins, including CDK6 and multiple MCMs were also significantly reduced when AR was activated by DHT. FKBP5, the immunophilin which negatively regulates proliferation and is thought to play various roles in the neoplastic process, was up-regulated by androgen ∼8 fold and ∼4 fold on the mRNA and protein levels, respectively. Collectively, these data provide a molecular basis for the decreased proliferation noted. Further, these data suggest that the expression of androgen/AR in thyroid cells plays a protective, anti-proliferative role in normal thyroid tissue that is lost in neoplastic transformation resulting in dysregulation of cell cycle genes normally under negative AR control. The expression levels of AR, as well as available androgens, may play a role in the etiology of PTC, the differential incidence of the disease, as well as the different clinical presentations and outcomes between women and men.

Citation Format: Melanie Elizabeth MacEwan, Timmy O'Connell, Hong Zhao, Codrin Iacob, Nina Suslina, Augustine Moscatello, Edward Shin, Raj Tiwari, Zbigniew Darzynkiewicz, Jan Geliebter. Androgen decreases proliferation of thyroid 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 2122. doi:10.1158/1538-7445.AM2014-2122
Abstract 2123: Her2 affects AR phosphorylation at serine 81 and suppresses cell proliferation of breast cancer cell line MDA-MB-453
Authors: Huang Pao-Hsuan , Huang Chen-Chuan , Lee Yueh-Tsung , Yue Chia-Herng , Lin Ho
Published: May 2015

Breast cancer is a hormone-related carcinoma and the most commonly diagnosed malignancy among women. Although human epidermal growth factor receptor-2 (Her2), estrogen receptor (ER), and progesterone receptor (PR) are the major diagnostic markers and therapeutic targets to breast cancer, searching for additional molecular targets becomes an important issue and one of the candidates is androgen receptor (AR). In recent studies, AR expresses in 70% breast cancer cases and its expression connects to low recurrence and high survival rate. Our previous study shows that S81 phosphorylation is critical for AR protein stability and modulated by Her2 in prostate cancer cells. In this study, we explore the roles of AR and Her2 in proliferation of breast cancer cell line, MDA-MB-453. The data show that AR which was activated by synthetic androgen R1881 decreased proliferation of MDA-MB-453 cells. AR knockdown increased both phosphorylation of Akt and ERK. Notably, AR activation decreased protein level of Early growth response 1 (Egr1) and increased protein level of cyclin-dependent kinase inhibitor p27, which suggests the correlation of AR activation and proliferation suppression. Besides, Her2 activation by Heregulin (HRG) decreased the levels of p-S81-AR. Her2 inhibition by small molecular inhibitor, Lapatinib, increased the levels of p-S81-AR and p27. In summary, our results indicate that Her2 might negatively regulate AR activation and contribute to suppressive proliferation of MDA-MB 453 cells.

Citation Format: Pao-Hsuan Huang, Chen-Chuan Huang, Yueh-Tsung Lee, Chia-Herng Yue, Ho Lin. Her2 affects AR phosphorylation at serine 81 and suppresses cell proliferation of breast cancer cell line MDA-MB-453. [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 2123. doi:10.1158/1538-7445.AM2014-2123
Abstract 2125: G-protein coupled receptors for cholecystokinin regulate transactivation of EGF receptors in lung cancer cells
Authors: Moody Terry W. , Lee Suk Hee , Perez Paola Moreno , Jensen Robert T
Published: May 2015

Cholecystokinin (CCK) receptors (CCK-AR and CCK-BR) are G-protein coupled receptors (GPCR) which are present on lung cancer cells (Yoder and Moody, Peptides 1987; 8: 103). CCK stimulates the proliferation of NCI-H345 cells, whereas the CCK-B receptor antagonist CI-988 inhibits proliferation (Moody et al., JPET 2001; 299: 1154). Peptide receptors for some gastrointestinal hormones/neurotransmitters mediate lung cancer growth by causing EGFR transactivation, but it is unknown if this occurs with CCK-BR. CCK-BR mRNA was detected in NCI-H345 and H727 lung cancer cells. Addition of CCK-8 or gastrin I (100 nM) to NCI-H345 or H727 cells, which have wild type EGFR, increased EGFR Tyr1068 phosphorylation after 5 min. The ability of CCK-8 to cause EGFR or ERK tyrosine phosphorylation was blocked by CI-988 (CCK-BR antagonist), gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), tiron (superoxide scavenger) and U-73122 (phospholipase C inhibitor) but not H89 (protein kinase A inhibitor). U-73122 or CI-988 inhibited the ability of CCK to cause phosphatidylinositol turnover and elevate cytosolic Ca2+. CI-988 or gefitinib inhibited the growth of NCI-H345 or H727 cells. CI-988 and gefitinib synergistically inhibited the growth of lung cancer cells. The results indicate that the CCK-BR regulates lung cancer proliferation in an EGFR-dependent mechanism through activation of Src, MMP and reactive oxygen species.

Citation Format: Terry W. Moody, Suk Hee Lee, Paola Moreno Perez, Robert T Jensen. G-protein coupled receptors for cholecystokinin regulate transactivation of EGF receptors in lung 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 2125. doi:10.1158/1538-7445.AM2014-2125
Abstract 2128: The effects of Quercetin on prostate cancer
Authors: Ward Ashley B. , Gupta Pranav , Kaur Gurpreet , Mir Hina , Lillard James W. , Singh Shailesh
Published: May 2015

Prostate cancer (PCa) affects nearly 80% of men worldwide and is the second leading killer after lung cancer. The efficacy of current treatments offered in the clinics is highly compromised due to indolent nature of PCa, which provides large window of opportunity for prevention. Hence, the major focus of this study is to determine the chemo-preventive effects of Quercetin, a bioflavonoid, on prostate cancer. Effect of Quercetin on cell viability and IC50 was determined by MTT assay. The effect of Quercetin on cell motility was determined by wound healing assay. Potential role of Quercetin on cell cycle, apoptosis as well as genes involved in cell motility and invasion was determined using flow cytometry, Real-time qPCR and ELISA. Furthermore, Quercetin induced changes in signaling molecules involved in cell survival/apoptosis, cell cycle and cytoskeletal rearrangement was determined using antibody microarray. Prostate cancer cells treated with Quercetin showed dose and time dependent inhibition of proliferation/viability, and induction of apoptosis as compared to normal prostatic epithelial cells and untreated controls. Prostate cancer cell motility was inhibited in Quercetin treated cells. Prostate cancer cells were arrested in G2 phase of the cell cycle following Quercetin treatment. In addition to these, we found differential expression of caspases, matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs in different PCa cell lines compared to untreated controls. Furthermore, antibody microarray analysis demonstrated selective modulation of genes and associated signaling cascades responsible for apoptosis induction, cellular motility, adhesion and invasion in Quercetin treated cells compared to controls. These findings suggest Quercetin as a potent chemo-preventive agent. In addition to this it can be also used with chemotherapeutic agents directed to G2 phase of the cell cycle, which may improve the efficacy of chemotherapeutics offered in clinics to treat advance prostate cancer.

Citation Format: Ashley B. Ward, Pranav Gupta, Gurpreet Kaur, Hina Mir, James W. Lillard, Shailesh Singh. The effects of Quercetin on prostate 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 2128. doi:10.1158/1538-7445.AM2014-2128