bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5326: MicroRNA profiles discriminate among colon cancer metastasis.
Authors: Acunzo Mario , Nuovo Jerry G. , Zanesi Nicola , Di Leva Giampiero , Pichiorri Flavia , Volinia Stefano , Fernandez Cecilia , Costinenan Stefan , Bottoni Arianna , Burch Aaron , Alder Hansjuerg , Conti Laura , Antenucci Anna , Rosito Immacolata A. , Ciardi Antonio , Liu Chang G. , Drusco Alessandra , Croce Carlo M.
Published: August 2014

MicroRNAs are being exploited for diagnosis, prognosis and monitoring of cancer and other diseases. Their small non immunogenic size, high tissue specificity, and critical role in oncogenesis provide new biomarkers for diagnosing and classifying cancer as well as predicting patients’ outcomes. MicroRNAs signatures have been identified for many human tumors, including colorectal cancer. This tumor affects about one million individuals in developed countries, and about half of them die for recurrences. In most cases, metastatic disease is difficult to predict and to prevent with adequate therapies. The identification of microRNAs signatures for metastatic disease, could aid to define the risk and localization of recurrences in patients with unknown micrometastasis.

In this study, tissues of three different groups of colorectal cancer (CRC) patients were analyzed: microarray and cards analysis were performed on 66 patients with or without lymph nodes and/or liver recurrences; a second group of 16 metastatic patients were selected for RT-PCR; finally, in situ hybridization was carried out on tissue cores arrays belonging to a different set of patients with and without positive lymph nodes and/or liver metastasis.

MicroRNA-31, 21, 93, and 103 up-regulation together with miR-566 down-regulation defined the CRC metastatic signature. Moreover, in situ hybridization data defined metastatic localization: miR-200c up-regulation together with miR-566 down-regulation characterized lynphonodal invasion, while liver recurrences were defined by miR-31, 93 and 103 over-expression.

Citation Format: Mario Acunzo, Jerry G. Nuovo, Nicola Zanesi, Giampiero Di Leva, Flavia Pichiorri, Stefano Volinia, Cecilia Fernandez, Stefan Costinenan, Arianna Bottoni, Aaron Burch, Hansjuerg Alder, Laura Conti, Anna Antenucci, Immacolata A. Rosito, Antonio Ciardi, Chang G. Liu, Alessandra Drusco, Carlo M. Croce. MicroRNA profiles discriminate among colon cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5326. doi:10.1158/1538-7445.AM2013-5326
Abstract 5334: Tumor-associated-macrophage induces CD44 expression through microRNAs suppression promotes cancer progression and chemoresistance.
Authors: Ishimoto Takatsugu , Watanabe Masayuki , Ozaki Nobuyuki , Iwatsuki Masaaki , Baba Yoshifumi , Yoshida Naoya , Baba Hideo
Published: August 2014

Objectives: CD44 has recently been identified as one of the cell surface markers associated with cancer stem cells (CSCs) in several types of tumor. We have reported the functional role of CD44 variant isoform in the maintenance of low reactive oxygen species (ROS) levels in gastrointestinal cancer cells. While, microRNA (miRNA) is involved in the pathogenesis of many cancers and the networks of miRNA regulate CSCs properties. However, the mechanism underlying the miRNA in the regulation of CSC marker is mostly unrevealed. Methods and Results: To investigate the mechanism in the regulation of CD44, we performed 384 miRNA quantitative RT-PCR array analysis in six human gastrointestinal cancer cell lines that differ in CD44 expression status. We found 59 miRNAs which are suppressed in CD44 high expressing cancer cells, and further investigated the predicted 33miRNAs which interact directly with CD44 3’UTR using the online miRNA target database. As a result in these analyses, we identified 2 miRNA candidates, which could regulate CD44 expression in gastrointestinal cancer. In this study, we especially focused on miR-328 and examined the functional relevance of miR-328 implicated in CD44 regulation. We found that gastrointestinal cancer cells transfected with miR-328 mimic decreased CD44 expression and led to inhibition of cancer cell growth and impairing chemo- and ROS- resistance. Furthermore, we investigated the mechanism underlying the regulation of miR-328 by using human macrophage and gastrointestinal cancer cell lines. We found that co-culture with tumor-associated-macrophages (TAMs) triggers the CD44 expression through suppression of miR-328 in gastrointestinal cancer cells and promote stem-like property that possess sphere-forming ability. Finally, we showed that TAMs are closely related with both CD44 over-expression and miR-328 under-expression in human gastric cancer tissue. Conclusions: These findings reveal that TAMs in tumor microenvironment contribute CD44 expression via miR-328 suppression, resulting in tumor progression through enhancing ROS defense.

Citation Format: Takatsugu Ishimoto, Masayuki Watanabe, Nobuyuki Ozaki, Masaaki Iwatsuki, Yoshifumi Baba, Naoya Yoshida, Hideo Baba. Tumor-associated-macrophage induces CD44 expression through microRNAs suppression promotes cancer progression and chemoresistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5334. doi:10.1158/1538-7445.AM2013-5334
Abstract 536: Insulin-like growth factor II (IGF-II) is a potential target for the treatment of triple negative breast cancers (TNBC).
Authors: Radhakrishnan Vinodh Kumar , Tan Qianwei , De León Marino A. , De León Daisy D.
Published: August 2014

In the United States, African American (AA) women have a lower incidence of breast cancer compared to Caucasian (CA) women, but higher overall mortality. Our lab have demonstrated that Breast Cancer from AA patients express significantly higher levels of IGF-II and show a higher activation of the IGF signaling system in African American (AA) when compared to Caucasian (CA) women. We showed that IGF-II promotes breast cancer progression and metastasis by inhibiting tumor cell death and promoting resistance to chemotherapy, thus contributing to increased breast cancer (BC) mortality. This study was designed to determine how IGF-II contributes to the aggressiveness of TNBC, the most deadly of breast tumors, which is highly prevalent among AA. To analyze IGF-II in a TNBC cell model, we chose cell lines established from tumors of AA and CA breast cancer patients. Expression of IGF-II and IGF-II regulated proteins mRNA expression was probed using the CFX96 real time PCR system. Similarly, IGF-II and related proteins expression was analyzed by western blots and chemiluminescence. We also characterized the epigenetic activation of IGF-II in these cell lines since variation in the expression of IGF-II alleles regulates a signature of proteins associated with tumor aggressiveness. Our exciting results demonstrated that higher IGF-II levels in TNBC cells from AA was associated with distinct epigenetic changes that included different promoter activation and higher levels of IGF-II translation which are inheritable traits. In contrast, TNBC from CA had higher IGF-II levels due to different molecular IGF-II regulation. Thus, IGF-II is an important biomarker and can be used as therapeutic target for treatment of TNBC.

Citation Format: Vinodh Kumar Radhakrishnan, Qianwei Tan, Marino A. De León, Daisy D. De León. Insulin-like growth factor II (IGF-II) is a potential target for the treatment of triple negative breast cancers (TNBC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 536. doi:10.1158/1538-7445.AM2013-536
Abstract 5374: Sulforaphane reactivates the expression of Nrf2 through CpGs demethylation in TRAMP C1 cells.
Authors: Zhang Chengyue , Su Zheng-Yuan , Khor Tin Oo , Kong Ah-Ng
Published: August 2014

Prostate cancer is one of the most deadly malignancies among men in the United States. Recent evidence suggests epigenetic alteration is involved during the development and progression of prostate cancer. Previously we have demonstrated that Nrf2, a key regulator of cellular antioxidant defense systems, was epigenetically silenced in TRAMP prostate cancers, as well as tumorigenic TRAMP C1 cells. Sulforaphane (SFN), an isothiocyanate rich in cruciferous vegetables, has been shown to be a potent cancer prevention agent for years. The aim of this study is to investigate the potential of SFN to reactivate the expression of Nrf2 in TRAMP C1 cells via epigenetic modifications. Bisulfite genomic sequencing (BGS) showed that treatment of SFN resulted in demethylation of the first 5 CpGs in the promoter region of Nrf2 gene. Methylation DNA immunoprecipitation (MeDIP) analysis revealed that SFN significantly reduced the anti-mecyt antibody binding to the first 5 CpGs of the Nrf2 promoter, corresponding to the BGS results. Reversion of the methylation status was found to be associated with re-expression of Nrf2 and its downstream NQO-1 at the mRNA and protein levels. Further analysis showed that SFN treatment decreased the expression of DNA methyltransferases (DNMTs), particularly DNMT1 and DNMT3a. Though SFN treatment showed differential effects on the protein expression of various HDACs, it increased the global level of active chromatin marker acetyl-H3. Taken together, our study suggests that SFN may exert its chemoprevention effect through epigenetic regulation modification of Nrf2 gene with subsequent induction of its downstream anti-oxidative stress pathway.

Citation Format: Chengyue Zhang, Zheng-Yuan Su, Tin Oo Khor, Ah-Ng Kong. Sulforaphane reactivates the expression of Nrf2 through CpGs demethylation in TRAMP C1 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5374. doi:10.1158/1538-7445.AM2013-5374
Abstract 538: IGF2 role in trastuzumab-resistance in JIMT-1 cells.
Authors: Helu Xousaen M. , De Leon Daisy D.
Published: August 2014

Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is detected in 20-30% of breast cancers. Overexpression of HER2 is linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity. Dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the insulin-like growth factor 1 receptor (IGF1R), the insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). We hypothesize that IGF2 mediates trastuzumab-resistance in JIMT-1 cell line. IGF2 mRNA and protein levels were determined in trastuzumab-sensitive cell lines (BT474, SKBR3, and ZR 75-30) and trastuzumab-resistant cell line (JIMT-1) by qPCR and Western blot. We also analyzed the effect of IGF2 inhibition treatment on the phosphorylation levels of HER2. Our study showed that IGF2 increases in trastuzumab-resistant cell line, JIMT-1. Furthermore, inhibition of IGF2 decreases JIMT-1 cell viability compared to control and renders cells sensitive to trastuzumab. These findings provide a potential therapy target for trastuzumab-resistant breast cancers.

Citation Format: Xousaen M. Helu, Daisy D. De Leon. IGF2 role in trastuzumab-resistance in JIMT-1 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2013-538
Abstract 5383: Overexpression of the vitamin D catabolizing enzyme CYP24A1 is caused by gene amplification and results in highly proliferative colorectal tumors.
Authors: Höbaus Julia , Aggarwal Abhishek , Hummel Doris M. , Thiem Ursula , Fetahu Irfete , Mesteri Ildiko , Kallay Enikö
Published: August 2014

Vitamin D insufficiency increases risk of colorectal cancer. Vitamin D is produced photochemically in the skin, thus, low sunlight exposure results in vitamin D insufficiency. Vitamin D is further processed in the liver to its storage form calcidiol (25-OH vitamin D3). Calcidiol can be activated by 1α-hydroxylation to the secosteroid hormone calcitriol (1α,25-OH vitamin D3). Although systemic levels of bioactive calcitriol are regulated by the kidneys, almost every tissue can synthesize and degrade calcitriol. Tissue calcitriol acts in an autocrine/paracrine manner and controls proliferation, apoptosis, and differentiation.

In colorectal cancer, the calcidiol and calcitriol-degrading enzyme CYP24A1 is substantially overexpressed both on mRNA and protein level. High CYP24A1 levels markedly reduce the half-life of vitamin D metabolites, likely reducing the anti-tumorigenic effects of calcitriol in the tumor. The causes and consequences of this overexpression are not fully understood.

Here, we investigated gene amplification of the CYP24A1 locus (20q13.2) as a possible cause of CYP24A1 overexpression and increased proliferation as a consequence thereof. Quantitative real time PCR assays showed that approximately 60% of colorectal tumors carry CYP24A1 gene amplification (n=127). This gene amplification correlated with increased mRNA expression (ρ=0.38, p<0.001). Aberrantly high CYP24A1 expression may reduce the anti-proliferative actions of calcitriol and lead to increased proliferation. In our colorectal cancer cohort, CYP24A1 mRNA expression correlated with expression of several proliferation markers (e.g. CDC6 ρ=0.60, p<0.001).

In conclusion, our data suggest that CYP24A1 gene amplification results in increased mRNA expression in colorectal tumors. Further, high CYP24A1 expression correlates with increased proliferation, possibly caused by an inhibition of the anti-proliferative effects of calcitriol. Tumor specific inhibition of CYP24A1 may provide a future strategy to restore local vitamin D levels and its anti-tumorigenic activities.

Citation Format: Julia Höbaus, Abhishek Aggarwal, Doris M. Hummel, Ursula Thiem, Irfete Fetahu, Ildiko Mesteri, Enikö Kallay. Overexpression of the vitamin D catabolizing enzyme CYP24A1 is caused by gene amplification and results in highly proliferative colorectal tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5383. doi:10.1158/1538-7445.AM2013-5383
Abstract 5386: Investigating the role of N-terminal protein methylation in colon cancer progression.
Authors: Tooley John G. , Trent John O. , Tooley Christine E. Schaner
Published: August 2014

We have recently identified the first mammalian N-terminal methyltransferase, NRMT. NRMT is a highly conserved, ubiquitously expressed nuclear enzyme that is predicted to methylate more than 300 target proteins, including the proven targets Retinoblastoma Protein (RB), DDB2, and the oncoprotein SET. N-terminal methylation has been shown to regulate protein-DNA interactions and has also been implicated in regulating protein stability. Multiple microarray analyses have shown that NRMT is overexpressed in human colon cancer, and our preliminary data indicate the more aggressive the cancer, the higher the NRMT expression. However, there are currently no commercially available inhibitors specific for this enzyme and very little is known about the downstream signaling consequences of NRMT misregulation. We have used in silico screening of the ZINC small molecule library to identify potential NRMT-specific inhibitors. Screening of the top 100 candidates produced 10 compounds that could inhibit NRMT methyltransferase activity in vitro. The top compound of these 10 can also inhibit NRMT methyltransferase activity in cell culture and specifically stops the growth of colon cancer cell lines that have high NRMT expression. We are currently working to optimize this small molecule inhibitor for stability and solubility, understand its downstream signaling consequences, and test its efficacy in a murine model system. We are especially interested in the potential of NRMT inhibitors as a treatment for patients with mutant kRAS. These patients are often insensitive to anti-EGFR therapy, but oncogenic RAS seems to require functional RB. Therefore, impairment of NRMT activity could also impair oncogenic RAS signaling through destabilization of RB. Data from this study will enhance our understanding of how colon cancer develops and could lead to novel treatments for the disease.

Citation Format: John G. Tooley, John O. Trent, Christine E. Schaner Tooley. Investigating the role of N-terminal protein methylation in colon cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2013-5386
Abstract 5424: CD44-mediated glucose metabolism affects ROS level and drug sensitivity in glycolytic cancer cells.
Authors: Tamada Mayumi , Suematsu Makoto , Saya Hideyuki
Published: August 2014

Low reactive oxygen species (ROS) level of cancer cells is implicated in chemo-resistance. One of the reasons why cancer cells keep their ROS level low is an increased glycolytic flux accompanied by activation of pentose phosphate pathway (PPP).

We found that CD44, a cell surface marker for cancer stem cells, interact with pyruvate kinase M2 (PKM2) which is one of the most important regulators of aerobic glycolysis (Warburg effect), and thereby promotes the glycolysis of cancer cells. CD44 ablation by RNA interference increased PKM2 enzymatic activity and then induced metabolic shift from glycolysis to mitochondrial respiration and concomitantly reduced glucose uptake and PPP flux via suppression of glucose transporter1 (Glut1) expression. Such metabolic changes by CD44 ablation depleted cellular reduced glutathione(GSH) contents and increased intracellular ROS level. Our group previously reported that CD44 variant expression promotes xCT-mediated cysteine uptake and consequent GSH synthesis. Taken together, we suggested that CD44 maintains GSH synthesis and low ROS level in glycolytic cancer cells not only through xCT-mediated cysteine uptake but also by maintenance of the PPP flux and consequent NADPH production.

Additionally, CD44 depleted cancer cells more sensitized them anticancer drugs even in more glycolytic environments, such as p53-deficient or hypoxia, in which cancer cells show more chemo-resistance. This increased sensitivity to anticancer drugs conferred by CD44 ablation was inhibited by pretreatment with a precursor of GSH that functions as an antioxidant. Therefore, these results indicated that enhanced ROS level by CD44 ablation sensitizes highly glycolytic cancer cells to conventional chemotherapy.

The possibility that CD44-targeted therapy may perturb the metabolism of cancer cells and thereby impair their capacity to defend against ROS warrants further investigation.

Citation Format: Mayumi Tamada, Makoto Suematsu, Hideyuki Saya. CD44-mediated glucose metabolism affects ROS level and drug sensitivity in glycolytic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5424. doi:10.1158/1538-7445.AM2013-5424
Abstract 5444: Role of CRD-BP in the pathogenesis of human basal cell carcinoma.
Authors: Noubissi Felicite Kamdem , Kim Tae Won , Kawahara Tisha , Longley B. Jack , Berg Eric , Athar Mohammad , Spiegelman Vladimir S.
Published: August 2014

Although the number of new cases of Basal Cell Carcinoma (BCC) has increased rapidly each year in the last few decades, the molecular basis of its pathogenesis is not completely understood. Activation of Hedgehog (Hh) signaling pathway was shown to be a key factor driving the development of BCC. The Wnt/β-catenin signaling pathway was also shown to be activated in BCCs and to perhaps modulate the activity of Hh pathway. We previously identified a novel mechanism by which Wnt signaling regulates the transcriptional outcome of Hh signaling pathway. We demonstrated that CRD-BP, a direct target of the Wnt/β-catenin signaling, induces the expression and transcriptional activity of the Hh transcriptional activator GLI1. We hypothesized that Wnt-induced and CRD-BP-dependent regulation of GLI1 expression and activities is important to the development of BCC. In this study, the role of CRD-BP in the development of human BCC will be discussed.

Citation Format: Felicite Kamdem Noubissi, Tae Won Kim, Tisha Kawahara, B. Jack Longley, Eric Berg, Mohammad Athar, Vladimir S. Spiegelman. Role of CRD-BP in the pathogenesis of human basal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5444. doi:10.1158/1538-7445.AM2013-5444
Abstract 5445: Differential roles of E2F1 in melanoma.
Authors: Meng Peng , Ghosh Rita
Published: August 2014

Melanoma, the malignant tumor of melanocytes, accounts for 4% of all skin cancer incidence but approximately 78% of skin cancer related deaths. No current treatment significantly enhances disease-free survival once distant metastases have established. The 5 year survival rate drops dramatically from 99.9% to 9% when melanoma progresses. To facilitate the development of effective treatment strategies, it is imperative that we obtain a better understanding of the biology of melanoma cells as they progress through the various stages of disease. Previous studies have shown that E2F1, a transcription factor, is over-expressed in melanoma cells. Interaction between E2Fs and tumor suppressor, Rb is essential for regulation of cell cycle progression. E2F1 is known to be involved in many other important biological processes, including DNA damage, DNA repair, differentiation, development, autophagy and apoptosis. Involvement of E2F1 in so many diverse functions underscores the importance of this protein under normal and pathologic conditions including melanoma. Previously we have found that primary melanoma cells have high level of E2F1 message and protein, and that the acetylation of E2F1 (active form) is higher in late stage melanoma cells. This led us to propose that E2F1 may function differentially in various stages of melanoma progression. Therefore we generated stable E2F1 knockdown cells in early stage primary and advanced metastatic melanoma cells. Our results show that E2F1 reduction works more effectively to inhibit cell growth in primary melanoma cells. Further, we also found differential effects on apoptosis and invasion in early and late stage melanoma cells. There is increased spontaneous cell death in primary melanoma cells after E2F1 knockdown, while there is increased apoptosis resistance in response to UV or valinomycin. These results taken together suggest that E2F1 has pleotropic effects in melanoma. It can switch between oncogenic or tumor suppressive functions during melanoma progression. A better understanding of the mechanism involved in this switch may allow combining gene therapies with other therapies for improved therapeutics for malignant melanoma. Supported by R21 CA125719 (RG).

Citation Format: Peng Meng, Rita Ghosh. Differential roles of E2F1 in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2013-5445
Abstract 5452: High-fat diet induces inflammation by increasing estrogen levels through Stat3, estrogen receptor alpha and aromatase in the mouse prostate.
Authors: Bhaskaran Natarajan , Shukla Sanjeev , Thakur Vijay S. , Babcook Melissa A. , MacLennan Gregory T. , Liu Guiming , Daneshgari Firouz , Gupta Sanjay
Published: August 2014

High-fat diet (HFD), an unfortunate lifestyle choice common in the Western world, is associated with inflammation and thereby is regarded as a risk factor for various diseases including obesity, diabetes, and cancer. The molecular mechanism(s) responsible for HFD-induced inflammation in the prostate gland are not well understood. Estrogen receptor alpha (ER-α), signal transducer and activator of transcription (Stat)-3 and aromatase are important signaling molecules constitutively activated during inflammation and have been shown to be upregulated in prostate cancer. We have previously reported that HFD activates a pro-inflammatory response in the prostate through elevated expression of Stat-3 (Prostate 72:233-43, 2012). In the current study, we sought to investigate a possible link between intraprostatic inflammation, HFD feeding, signaling molecules involved in inflammation viz. ER-α, Stat-3, p-Stat3 (Ser727) and aromatase. C57BL/6 mice were fed either a regular diet (RD) or a HFD for 4, 8 and 12 weeks. Serum, visceral fat and prostate tissues were obtained for analysis. HFD intake caused an increase in fat and serum testosterone levels. No significant changes were observed in the testosterone levels in the prostate. A marked increase in estrogen levels was noted in the prostate and visceral fat at 8 weeks of HFD intake. HFD feeding resulted in a significant increase in the expression of aromatase, ER-α, Stat-3 and p-Stat3 (Ser727) at the message and protein levels. Immunoprecipitation and ChIP analysis demonstrated an increased association between aromatase, p-Stat-3 and ER-α in the HFD group. IHC analysis revealed higher expression of Stat-3, ER-α and aromatase in the prostate, accompanied by the morphologic evidence of increased intraprostatic inflammation in the HFD group. Taken together, our findings suggest that HFD increases estrogen levels by increased binding of ER-α and p-Stat-3 to the promoter of aromatase and their interaction is associated with increased intraprostatic inflammation which might contribute to the initiation and development of prostate cancer.

Citation Format: Natarajan Bhaskaran, Sanjeev Shukla, Vijay S. Thakur, Melissa A. Babcook, Gregory T. MacLennan, Guiming Liu, Firouz Daneshgari, Sanjay Gupta. High-fat diet induces inflammation by increasing estrogen levels through Stat3, estrogen receptor alpha and aromatase in the mouse prostate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5452. doi:10.1158/1538-7445.AM2013-5452
Abstract 5454: Androgen-independent regulation of cellular signaling, transcriptional activity, and mRNA stability in a cell model of prostate cancer.
Authors: Smith Suzanne M. , Opperman Kay K. , Kaboord Barbara J. , Bell Peter A.
Published: August 2014

Prostate cancer progression relies on the interplay between androgen dependent and independent processes. Depending on the cell type and stimulus, the independent pathway may result in proliferation, progression, or inhibition. It recently has become evident that the molecular mechanisms that drive the differentiation of normal prostate cells towards abnormal proliferation involve multiple signal transduction cascades that often overlap and interact. Through a combination of multiple techniques, including chromatin immunoprecipitation (chIP) and RNA pull-down, we are able to correlate cellular signaling events to androgen receptor transcriptional activity, and how it relates to cancer progression by using growth factors and hormones. In growth factor stimulated LNcap prostate cancer cells, we observed activation of Stat3 and PI3K pathways and reduced transcriptional activity of typical androgen receptor DNA targets, such as prostate-specific antigen (PSA) and cyclin-dependent kinase inhibitor (CDKN1A or p21). However, in estradiol stimulated cells we detected an increase transcriptional activity of the above targets as well as activation of proliferation pathways. Because the androgen receptor and its targets are also regulated by mRNA stability through the UTR, we assessed RNA stability using RNA pull-down assays and RT-PCR. Our results, and those already published, indicate that multiple overlapping signal transduction pathways are involved in the abnormal differentiation of prostate cells; therefore, our use of a combination of tools is advantageous in further defining the model of cancer progression and potential therapies.

Citation Format: Suzanne M. Smith, Kay K. Opperman, Barbara J. Kaboord, Peter A. Bell. Androgen-independent regulation of cellular signaling, transcriptional activity, and mRNA stability in a cell model of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5454. doi:10.1158/1538-7445.AM2013-5454
Abstract 5457: FOXO3a transcriptionally regulates pro-apoptotic function of Par-4 and induces apoptosis in prostate cancer cells.
Authors: Das Trinath P. , Damodaran Chendil
Published: August 2014

Deletion and mutation of tumor suppressor genes (PTEN and p53) may play an important role in the carcinogenesis of prostate cancer (CaP). Constitutive activation of Akt promotes cell proliferation and survival by regulating a number of transcription factors/genes in many cancer cells including CaP. Activation of AKT negatively regulates the pro-apoptotic functions of Fork head transcription factor (FOXO3a) and prostate apoptosis response-4 (Par-4) in CaP cells. In our studies, we found a molecular interplay between FOXO3a and Par-4 in CaP cells and more precisely, how, FOXO3a regulates Par-4 function in CaP cells. Knockin and knockout assays in CaP cells suggest that FOXO3a is upstream of Par-4 and FOXO3a may responsible for the transactivation of Par-4 and its pro-apoptotic function in CaP cells. Further, Immunofluorescence assays suggest during apoptotic stimuli, Par-4 and FOXO3A colocalization in nucleus, which in turn initiates pro-apoptotic signaling and induction of apoptosis. These results were further confirmed by pull down assays, which supports the physical interaction of these two proteins. These findings encouraged us to explore the FOXO3a binding sites in the promoter region of Par-4. We identified four FOXO3a binding sites at -2075 to -2069, -1810 to -1804, -1561 to -1555, and -1340 to -1334 in the promoter region of Par-4 gene. Luciferase reporter constructs of Par-4 promoter (2.1 kb) spanning the FOXO3a binding sites and sequentially deleted constructs were utilized to identify the crucial FOXO3a binding site for the regulation of Par-4. Sequential deletion of FOXO3a binding sites in Par-4 promoter resulted in inhibition of Par-4 function and induction of apoptosis in CaP cells. Further, confirm the role of FOXO3a, we specifically activated Par-4 function by small molecules and simultaneously co-transfect with dominant-negative FOXO3a constructs. As expected dominant negative FOXO3a abrogates Par-4 function in CaP cells, confirming FOXO3a is an upstream of Par-4 signaling and it is required for Par-4 induced apoptosis in CaP cells. We believe, these results decipher the mechanisms associated with FOXO3a induced Par-4 gene activation and apoptosis in CaP cells and activating of FOXO3a may result Par-4 mediated apoptosis in CaP cells.

Citation Format: Trinath P. Das, Chendil Damodaran. FOXO3a transcriptionally regulates pro-apoptotic function of Par-4 and induces apoptosis in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2013-5457

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 546: GDC-0032 PI3K inhibitor enhances the efficacy of endocrine therapies in breast cancer cells by differentially regulating the expression of ER-targeted genes and increasing apoptosis.
Authors: Wallin Jeffrey J. , Guan Jane , Hong Rebecca , O'Brien Carol , Wilson Timothy , Nannini Michelle , Sampath Deepak , Belvin Marcia , Friedman Lori S.
Published: August 2014

Endocrine therapies such as letrozole or fulvestrant are commonly used treatment options for metastatic Hormone Receptor positive (HR+) breast cancer but patients ultimately relapse. Phosphatidylinositol 3-kinases (PI3K) regulate breast tumor cell growth, migration and survival. The alpha isoform of PI3K is frequently mutated and activated in HR+ breast cancer and has been implicated in resistance to endocrine therapies. PI3K inhibitors are therefore attractive for combination with endocrine therapies. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that GDC-0032 has increased activity against PI3K alpha isoform (PIK3CA) mutant and HER2-amplified cancer cell lines. Single agent and combination studies were carried out to determine if GDC-0032 enhances the anti-tumor activity of endocrine therapies in human breast cancer models. MCF7 cells (Estrogen Receptor positive (ER+), PI3K alpha E545K mutant) were transfected with the aromatase gene and stable clones (MCF7-ARO) were selected that are capable of converting androstenedione to estrogen in culture. When grown in the presence of androstenedione, MCF7-ARO cells were more reliant on estrogen for growth. Under these conditions the cells were treated with GDC-0032 in combination with endocrine therapies and assayed for cellular viability, modulation of PI3K pathway and ER pathway markers and apoptosis induction. The combination of GDC-0032 and endocrine therapies decreased the cellular viability of MCF7-ARO cells and increased apoptosis relative to either single agent. We observed cross-talk between the PI3K and ER pathways that suggests a mechanism of action for the combination. The data provide rationale for evaluating GDC-0032 in combination with endocrine therapies for HR+ breast cancer treatment in the clinic.

Citation Format: Jeffrey J. Wallin, Jane Guan, Rebecca Hong, Carol O'Brien, Timothy Wilson, Michelle Nannini, Deepak Sampath, Marcia Belvin, Lori S. Friedman. GDC-0032 PI3K inhibitor enhances the efficacy of endocrine therapies in breast cancer cells by differentially regulating the expression of ER-targeted genes and increasing apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 546. doi:10.1158/1538-7445.AM2013-546
Abstract 5538: Direct inhibition of ERK1/2 by VTX-11e leads to increased induction of apoptosis in a subset of pancreatic cancer cell lines as compared to MEK1/2 inhibition by selumetinib (AZD6244).
Authors: Miermont Anne M. , Pflicke Holger , Guan Hannah , Chinnasamy Harshini , Thomas Craig , Rudloff Udo
Published: August 2014

Background: Intense efforts to improve current molecular therapy in pancreatic ductal adenocarcinoma (PDAC) have focused on suppression of the MAPK/ERK1/2 pathway. Inhibitors of MEK1/2, downstream target of the frequently mutated RAS oncogene, are currently in development. Recently, results of a phase II clinical trial revealed that selumetinib, a second-generation MEK1/2 inhibitor, has not yielded its promising preclinical efficacy in PDAC patients. A novel Extracellular Signal Regulated Kinase 1/2 (ERK1/2) inhibitor, VTX-11e, has been recently developed as a potent and selective inhibitor of ERK1/2. In this study, we evaluated the effects of VTX-11e on cell growth and apoptosis in pancreatic cancer cell lines and compared VTX-11e to selumetinib.

Methods: Fifty percent Growth Inhibition (GI50) and Lethal Dose (LD) were determined at 72 hours in 65 PDAC cell lines with increasing concentrations of VTX-11e or selumetinib. Apoptosis was quantified by Annexin V/PI following VTX-11e or selumetinib treatment at increasing concentrations at 24 and 72 hours and 7 days. Elk1 phosphorylation levels were determined by Western Blotting as a well-established readout for downstream MAPK pathway output.

Results: More than a third of VTX-11e-treated PDAC lines showed GI50 concentrations of ≤2μM. LD drug response curves of cell lines treated with VTX-11e showed cell death induction at lower concentrations than selumetinib in close to half the cell lines tested. This finding was confirmed by assessment of apoptosis by the AnnexinV/PI assay. We demonstrated that VTX-11e induced significant increased apoptosis in a subset of the PDAC lines compared to selumetinib, leading to as much as 50% apoptotic cells. Furthermore, VTX-11e and selumetinib suppressed phosphorylation of downstream target Elk1 equally.

Conclusion: Our results show that the novel ERK1/2 inhibitor VTX-11e leads to a significant increase in apoptosis as compared to the MEK1/2 inhibitor selumetinib in a subset of pancreatic cancer cell lines. This finding suggests that targeting ERK1/2 directly may be a more effective therapeutic strategy than targeting upstream MEK1/2 in some PDAC. Current studies are ongoing to determine the differential mechanism of action between VTX-11e and selumetinib.

Citation Format: Anne M. Miermont, Holger Pflicke, Hannah Guan, Harshini Chinnasamy, Craig Thomas, Udo Rudloff. Direct inhibition of ERK1/2 by VTX-11e leads to increased induction of apoptosis in a subset of pancreatic cancer cell lines as compared to MEK1/2 inhibition by selumetinib (AZD6244). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5538. doi:10.1158/1538-7445.AM2013-5538
Abstract 5552: Manganese porphyrins in combination with ascorbate act as pro-oxidants and mediate caspase-independent cancer cell death.
Authors: Evans Myron , Tovmasyan Artak , Batinic-Haberle Ines , Devi Gayathri
Published: August 2014

Resistance to therapy-mediated apoptosis in inflammatory breast cancer (IBC), an aggressive and distinct subtype of breast cancer was recently attributed to increased antioxidant expression (superoxide dismutase [SOD]; reduced glutathione) and decreased accumulation of reactive oxygen species (ROS) resulting in redox adaptation and increased cell survival. Mn porphyrins (MnPs) are amongst the most potent synthetic mimics of the antioxidant, superoxide dismutase (SOD). In the present study, we demonstrate the unique ability of two Mn porphyrin (MnP)-based SOD mimics (MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+) to act as pro-oxidants when combined with ascorbate (vitamin C) and enhance ROS-mediated cell death. We identified that the ethyl species, MnTE-2-PyP5+, was less stable than its butoxyethyl analog, MnTnBuOE-2-PyP5+, resulting in greater potency in inducing cytotoxicity via radical-driven oxidative degradation in cellular models of IBC isolated from patient tumors and an isogenic derivative with acquired resistance to therapeutic apoptosis. The combination of MnP and ascorbate induced accumulation of mitochondrial superoxide and H2O2-derived radicals in both therapy-sensitive and therapy-resistant cell lines. High intracellular ROS resulted in decreases in both pro-survival signaling (pNF-κB, pERK1/2) and expression of X-linked inhibitor of apoptosis protein (XIAP), the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and Annexin V staining, administration of a pan-caspase inhibitor (QVD-OPh) did not reverse the observed cytotoxicity. Nuclear translocation of apoptosis inducing factor (AIF) was observed, suggesting a caspase-independent cell death following mitochondrial outer membrane permeabilization after treatment with this combination. In conclusion, this study provides the molecular basis of a novel MnP-based anti-cancer strategy and suggests the feasibility of using MnPs to sensitize cancer cells to many chemotherapies and radiotherapy that are dependent on potent induction of the oxidative stress response.

Citation Format: Myron Evans, Artak Tovmasyan, Ines Batinic-Haberle, Gayathri Devi. Manganese porphyrins in combination with ascorbate act as pro-oxidants and mediate caspase-independent cancer cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5552. doi:10.1158/1538-7445.AM2013-5552

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 556: Cot/Tpl2 is required for neoplastic cell transformation induced by interleukin-22: a possible mechanism in breast carcinogenesis.
Authors: Kim Karam , Choi Hong Seok
Published: August 2014

Interleukin 22 (IL-22), a member of the IL-10 family of cytokines, is secreted T helper 17 (Th17) cells. Although IL-22 was recently reported to be a novel inflammation mediator through STAT3 signaling activation, the precise mechanisms of IL-22 in neoplastic cellular transformation and breast carcinogenesis are not well explored yet. Here, we report that IL-22 induced neoplastic transformation of JB6 Cl41 cells through activation of Cot/Tpl2. IL-22 dose- and time-dependently increased Cot/Tpl2 phosphorylation in JB6 Cl41 cells through IL-22 receptor. IL-22 phosphorylated MEK1/2, JNK1/2 and STAT3, which were inhibited by a specific inhibitor of Cot/Tpl2 kinase. When small interfering RNA (siRNA) of IL-22 and IL-22 receptor (IL-22R) were transfected into JB6 Cl41 cells, IL-22-induced Cot/Tpl2 phosphorylation was markedly decreased, followed by inhibition of MEK-ERK, JNK-c-Jun and STAT3 signaling pathway. Also, IL-22 activated c-fos and c-jun promoter activity, leading to increased activator protein-1 (AP-1) activity, which was suppressed by Cot/Tpl2 inhibitor. The in vivo chorioallantoic membrane (CAM) assay also showed that IL-22 increased tumor growth of JB6 Cl41 cells, whereas Cot/Tpl2 inhibitor suppressed the tumorigenesis induced by IL-22. In addition, knockdown of IL-22 and/or inhibition of Cot/Tpl2 diminished tumorigenecity of human breast cancer SKBR3 cells. Consistent with these observations, immunohistochemical staining showed that both expressions of IL-22 and Cot/Tpl2 proteins were highly upregulated in human breast cancers. Thus, this suggests that Cot/Tpl2 plays a pivotal role in IL-22-induced neoplastic cell transformation, and that Cot/Tpl2 may be the therapeutic target of breast cancer promoted by IL-22.

Citation Format: Karam Kim, Hong Seok Choi. Cot/Tpl2 is required for neoplastic cell transformation induced by interleukin-22: a possible mechanism in breast carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 556. doi:10.1158/1538-7445.AM2013-556

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 5596: Linkage between the lipogenic pathway and the progression of breast carcinomas.
Authors: Gonzalez-Guerrico Anatilde M. , Park-Hong Cheol , Kleer Celina , Espinoza Ingrid , Lupu Ruth
Published: August 2014

Fatty acid synthase (FASN) is a lipogenic enzyme responsible for the novo synthesis of fatty acids (FAs) from acetyl-CoA and malonyl-CoA in a NADPH dependent manner in cells. FAs are essential constituents of all biological membranes lipids and are important substrates for energy metabolism. While normal cells (except liver and adiposities) express low levels of FASN and have low to undetectable levels of FASN enzymatic activity, FASN expression and activity is observed in a vast majority of human cancers. Whether the increase in FASN is merely a consequence of disease progression or it is a “metabolic oncogene” that drives disease progression remains unclear. Here, we undoubtedly demonstrated that knockdown FASN results in inhibition of tumor formation and angiogenesis in athymic nude mice. Moreover, the histopathological features of the FASN knockdown remaining tissue in vivo appear globular, well differentiated with low nuclear grade compared to the control tumors, which appear invasive, undifferentiated, and of high nuclear grade. When tissues where stained for Ki-67, Vimentin and E-Cadherin the classical mesenchymal to epithelial transition (MET) was observed in the tissue derived from FASN knockdown tumors. Our studies establish a possible role for FASN as a “metabolic oncogene” in breast cancer. Given the data presented here, we propose that FASN is a perfect marker to develop personalized targeted therapy.

Citation Format: Anatilde M. Gonzalez-Guerrico, Cheol Park-Hong, Celina Kleer, Ingrid Espinoza, Ruth Lupu. Linkage between the lipogenic pathway and the progression of breast carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5596. doi:10.1158/1538-7445.AM2013-5596