bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1251: Repurposing antipsychotic drug Penfluridol for cancer treatment
Authors: Ranjan Alok , Srivastava Sanjay K.
Published: May 2015

Repurposing of old drugs as new anti-cancer drugs is important as it can save time and cost of drug development. Penfluridol is a first generation, highly potent antipsychotic drug. In this study, we determined the anticancer effects of Penfluridol in pancreatic cancer cells. Penfluridol treatment inhibited the growth of Panc-1, BxPC-3 and AsPC-1 pancreatic cancer cells in a concentration-dependent manner. Panc-1 , BxPC-3 and AsPC-1 cells treated with Penfluridol exhibited apoptosis as evaluated by Annexin/FITC assay and cleavage of caspase-3 and PARP. Our results showed that Penfluridol treatment induced ER stress in pancreatic cancer cells through the up regulation of ER stress markers like Bip/Grp78, CHOP and IRE. Penfluridol treatment also induced autophagy in pancreatic cancer cells as observed by acridine orange assay. Western blot analysis of Panc-1, BxPC-3 and AsPC-1 cells treated with Penfluridol exhibited up regulation of autophagy markers like LC3B, and p62. Microscopic analysis revealed punctate LC3B after Penfluridol treatment in pancreatic cancer cells confirming autophagy. Further mechanistic studies are in progress to establish the role of ER stress and autophagy in pancreatic cancer cells and correlate it with cell growth suppression. [Supported in part by R01 grant CA129038 awarded by NIH.]

Citation Format: Alok Ranjan, Sanjay K. Srivastava. Repurposing antipsychotic drug Penfluridol for cancer treatment. [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 1251. doi:10.1158/1538-7445.AM2014-1251
Abstract 1371: Histone lysine trimethylation or acetylation in sporadic breast tumor and matched normal tissue
Authors: Gallon-Bernard Dominique J. , Judes Gaëlle , Dagdemir Aslihan , Echegut Maureen , Karsli-Ceppioglu Seher , Ngollo Marjolaine , Lebert Andre , Penault-Llorca Frederique , Bignon Yves-Jean
Published: May 2015

Breast cancer is responsible of cancer mortality in women worldwide. The challenge in carcinogenesis is to predict tumor progression. Epigenetics open new perspectives in this field. It was demonstrated that epigenetic alterations are involved in cancer. DNA methylation or histone modifications can regulate gene transcription. Our goal was to determine the role of different histone marks, such as H3K27me3, H3K4ac and H3K9ac on the expression of a panel of genes involved in breast cancer: BRCA1, ERβ, EZH2, P300, SRC3, ERα and PGR. For this study, we used less than one hundred sporadic breast tumors and matched normal tissues. We performed chromatin immunoprecipitation and Q-PCR. After that, we checked the gene expression assaying RNA with RT-QPCR and proteins with Western blotting. Finally, we demonstrated that H3K4ac marks behaved like H3K27me3 repressive marks. The increase of H3K9ac marks resulted in the overexpression of ERα and P300 genes in the tumors. For the underexpressed genes BRCA1 and ERβ, we found that their repression did not pass through the repressive marks H3K27me3 nor H3K4ac but by other mechanisms perhaps DNA methylation.

Then, we used variance analysis and studied interactions between the studied different marks and intrinsic subtypes of breast cancer according to St Gallen International Expert Consensus. Finally, we defined gene-mark couples in each subtypes. At the end, a decision tree was drawn and determined 4 genes (BRCA1, ERα, ERβ, and PGR) and 2 marks (H3K27me3 and H3K4ac) that separated subtypes in the present study.

Citation Format: Dominique J. Gallon-Bernard, Gaëlle Judes, Aslihan Dagdemir, Maureen Echegut, Seher Karsli-Ceppioglu, Marjolaine Ngollo, Andre Lebert, Frederique Penault-Llorca, Yves-Jean Bignon. Histone lysine trimethylation or acetylation in sporadic breast tumor and matched normal tissue. [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 1371. doi:10.1158/1538-7445.AM2014-1371
Abstract 1390: Interferon-gamma potentiates the targeted phenotypic reversion of erbb2/her2/neu transformed human tumor cells
Authors: Runkle Edwin Aaron , Young Peter , Nagai Yasuhiro , Tsuchiya Hiromichi , Zhang Hongtao , Greene Mark I.
Published: May 2015

Oncogenic erbB2/HER2 is a major transforming determinant of human breast cancer and its amplification is prognostic of poor survival. Monoclonal antibody (mAb)-based therapy to specifically target erbB2/HER2 has been effective in patients with HER2-positive breast cancer; however, some patients do not respond and those who do frequently relapse despite ongoing therapy. The evolution of tumors that resist targeted therapy is complex and involves the emergence of complex transcriptional functions. Snail is overexpressed in recurrent tumors of transgenic HER2/neu mice and snail expression participates in tumor recurrence. Previous studies from our laboratory and others have revealed that targeted therapy requires cooperation with elements of the endogenous immune system for optimal tumor regression activity. The cytokine interferon-gamma (IFN-γ) is relevant to the regression process although the mechanism by which IFN operates on targeted cells remains undefined and the effects of IFN on the transformed cell have not been studied during phenotypic reversion. In the present study, we utilized cell biological and biochemical-based assays to define if targeted therapy could be improved through the addition of IFN-γ and unexpectedly defined a unique mechanism operative to achieve this end. Cell culture model systems were used to examine the co-treatment of erbB2/HER2 targeting with IFN-γ to eliminate issues such as aberrant immune responses, which arise as a consequence of adding IFN-γ to cells. We defined that while mAb targeting of erbB2 reversed the transformed phenotype of HER2-positive breast cancer cells reversion of phenotype was increased upon inclusion of IFN-γ. Co-treatment with the erbB2 mAb and IFN-γ resulted in a greater reduction of snail protein levels compared to either modality alone. Snail degradation was dependent on GSK3-β and active proteasome dependent elimination processes. Furthermore, IFN-γ activated GSK3-β in mAb treated cells to the extent of that observed in cells treated with specific small molecule inhibitors to erbB receptors. Our findings provide a mechanistic explanation as to how to use IFN-γ to optimize mAb-mediated tumor regression. The implication of this study is improvement of targeted efficacy could lead to more comprehensive therapy options that may eliminate tumor recurrence.

Citation Format: Edwin Aaron Runkle, Peter Young, Yasuhiro Nagai, Hiromichi Tsuchiya, Hongtao Zhang, Mark I. Greene. Interferon-gamma potentiates the targeted phenotypic reversion of erbb2/her2/neu transformed human tumor 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 1390. doi:10.1158/1538-7445.AM2014-1390
Abstract 140: The role of Abl kinases in breast cancer bone metastasis
Authors: Wang Jun
Published: May 2015

Abl kinases are a family of non-receptor tyrosine kinases consisting of two members, Abl1 and Abl2 (Arg). In recent years, accumulating data support a role of Abl kinases in breast cancer that harbor enhanced expression and/or activation of these kinases. The Cancer Genome Atlas has recently reported that Abl2 is among the most highly amplified genes and overexpression of Abl2 correlates with poor survival in invasive breast cancer. In addition, Abl kinases are activated downstream of receptor tyrosine kinases, chemokine receptors and src family kinases, all of which are deregulated in breast cancer and drive tumor proliferation and invasion. In this study, we have uncovered a critical role of Abl kinases in the regulation of breast cancer metastasis to the bone. We found that the incidence of bone metastasis is significantly greater in breast cancer patients with high Abl expression. Using global transcriptome analysis (RNAseq) of breast cancer cells we uncovered a novel role for Abl kinases in regulating the expression of multiple pro-bone metastasis genes. Furthermore, both pharmacological inhibition and RNA interference mediated knockdown of Abl kinases resulted in markedly decreased osteolytic metastasis in vivo and decreased osteoclast activation in vitro. Together these findings support a role for Abl kinases in promoting osteolytic metastasis and suggest that Abl dependent signaling may be targeted to inhibit this process.

Citation Format: Jun Wang. The role of Abl kinases in breast cancer bone metastasis. [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 140. doi:10.1158/1538-7445.AM2014-140
Abstract 1406: Evoking potent RNAi response using novel 2′-OMe-phosphorodithioated modified siRNAs
Authors: Wu Sherry , Yang Xianbin , Egli Martin , Ghaupure Kshipra , Hatakeyama Hiroto , McGuire Michael , Rupaimoole Rajesha , Miyake Takahito , Taylor Morgan , Pradeep Sunila , Nagaraja Archana , Sierant Malgorzata , Singhania Richa , Rodriguez-Aguayo Cristian , McMillan Nigel , Lopez-Berestein Gabriel , Ram Prahlad , Nawrot Barbara , Sood Anil K.
Published: May 2015

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a critical challenge to bringing siRNA therapy into the clinic. There is currently an unmet need to develop a reliable strategy to globally enhance siRNA stability and potency. Here, we report a novel chemical modification, consisting of phosphorodithioate (PS2) and 2′-O-Methyl (2′-OMe) MePS2 on a single nucleotide, that significantly enhances potency and resistance to nuclease degradation for a variety of siRNA sequences. We show a 3.5-fold improvement in gene silencing in tumors following systemic delivery of MePS2-modified siRNAs using DOPC nanoliposomes compared to unmodified counterparts. We found that this enhanced potency stems from an unforeseen increase in loading of siRNAs to the RNA-induced silencing complex (RISC), likely due to the unique interaction between 2′-OMe and PS2 moieties. We subsequently demonstrate the therapeutic utility of MePS2 siRNAs in orthotopic mouse models of chemoresistant ovarian cancer, focusing on targeting GRAM domain containing 1B (GRAMD1B), a protein whose role in taxane resistance is first reported here. Efficient silencing of GRAMD1B (>80%) was achieved in tumors following systemic delivery of these MePS2-modified siRNAs and a synergistic anti-tumor effect was observed when combined with paclitaxel treatment. Given that limited success has been achieved thus far toward broadly enhancing siRNA potency with chemically modified siRNAs, our finding represents an important step forward in bringing siRNA therapeutics into the clinic.

Citation Format: Sherry Wu, Xianbin Yang, Martin Egli, Kshipra Ghaupure, Hiroto Hatakeyama, Michael McGuire, Rajesha Rupaimoole, Takahito Miyake, Morgan Taylor, Sunila Pradeep, Archana Nagaraja, Malgorzata Sierant, Richa Singhania, Cristian Rodriguez-Aguayo, Nigel McMillan, Gabriel Lopez-Berestein, Prahlad Ram, Barbara Nawrot, Anil K. Sood. Evoking potent RNAi response using novel 2′-OMe-phosphorodithioated modified siRNAs. [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 1406. doi:10.1158/1538-7445.AM2014-1406
Abstract 1438: Targeting metabolic enzyme with locked nucleic acids in non-small cell lung cancer
Authors: Zhang Wen Cai , Lim Bing
Published: May 2015

Despite advances in our knowledge of genetics of cancer and some progress in cancer treatment over the last few decades, lung cancer remains the leading cause of cancer mortality worldwide. Recently cancer metabolomics have been widely applied into cancer research, including of cancer diagnosis and therapy. Our recent work has identified tumor initiating cells (TICs) derived from non-small cell lung cancer (NSCLC) patients and shown that metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in NSCLC. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients. Those findings provide potential diagnostic markers and/or therapeutic target for NSCLC. Locked nucleic acids (LNAs) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. In this study we tested NSCLC patient-derived xenograft tumor growth inhibition by GLDC LNAs antisense oligonucleotides. These LNAs antisense oligonucleotides strongly reduce GLDC protein levels. The osmotic minipumps were filled with GLDC LNAs antisense oligonucleotides at 0.5,1 and 5 mg/kg/day dose and implanted dorsally into tumor-bearing NOD/SCID Il2rγ-/- mice. Tumor growth was inhibited at dosages from 1 mg/kg/day. LNAs antisense oligonucleotides appeared to be non-toxic at dosages of 5 mg/kg/day. Furthermore, we found significant changes of serum metabolomics in lung cancer patients compared to normal donors. Our data show that GLDC LNAs antisense oligonucleotides are potent genotype-specific drugs that can inhibit lung tumor growth in vivo.

Note: This abstract was not presented at the meeting.

Citation Format: Wen Cai Zhang, Bing Lim. Targeting metabolic enzyme with locked nucleic acids in non-small cell 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 1438. doi:10.1158/1538-7445.AM2014-1438
Abstract 1474: Integrated microRNA network analyses identify a poor prognosis subtype of gastric cancer characterized by the miR-200 family
Authors: Song Fengju , Chen Kexin
Published: May 2015

Purpose:

Our aim was to investigate whether microRNAs can predict the clinical outcome of patients with gastric cancer (GC). We used integrated analysis of microRNA and mRNA expression profiles to identify GC microRNA subtypes and their underlying regulatory scenarios.

Experimental Design:

MicroRNA-based GC subtypes were identified by consensus clustering analysis of microRNA profiles of 90 GC tissues. Activated pathways in the subtypes were identified by gene expression profiles. Further integrated analysis was performed to model a microRNA regulatory network for each subtype. RNA and protein expression were analyzed by RT-PCR and tissue microarray, respectively, in a cohort of 385 GC cases (including the 90 cases for profiling) to validate the key microRNAs and targets in the network. Both in vitro and in vivo experiments were performed to further validate the findings.

Results:

MicroRNA profiles of 90 GC cases identified two microRNA subtypes significantly associated with survival. The poor-prognosis GC microRNA subtype was characterized by overexpression of epithelial-to-mesenchymal transition (EMT) markers. This GC “mesenchymal subtype” was further validated in a patient cohort comprising 385 cases. Integrated analysis identified a key microRNA regulatory network likely driving the GC mesenchymal subtype. Three of the microRNAs (miR-200c, miR-200b, and miR-125b) targeting the most genes in the network were significantly associated with survival. Functional experiments demonstrated that miR-200b suppressed ZEB1, augmented E-cadherin, inhibited cell migration, and suppressed tumor growth in a mouse model.

Conclusions: We have uncovered a key microRNA regulatory network that defines the mesenchymal GC subtype significantly associated with poor overall survival in GC.

Citation Format: Fengju Song, Kexin Chen. Integrated microRNA network analyses identify a poor prognosis subtype of gastric cancer characterized by the miR-200 family. [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 1474. doi:10.1158/1538-7445.AM2014-1474
Abstract 1483: Biological and clinical significance of miR-224 in colorectal cancer
Authors: LING HUI , Calin George , Nicoloso Milena , Ikuo Mariko
Published: May 2015

MicroRNAs (miRNAs) not only participate to cancer initiation and tumor growth, but also play roles in tumor metastasis. The aims of this study were to identify specific miRNAs involved in colorectal cancer (CRC) metastasis, and study their biological functions. We initially assessed by microarray global miRNA expression profile in primary CRC tissues from patients with or without metastasis. The most differentially expressed miRNAs were validated in a large set of CRC patient samples by real time PCR (qRT-PCR). We identified 7 miRNAs that were significantly upregulated in stage III-IV primary CRC compared with stage I-II cancer, among which miR-224 expression increased consistently with tumor burden. We used transwell-based cell motility analysis in CRC cell lines to test the biological activities of studied miRNAs, and showed that miR-224 and miR-141 promote CRC cell motility. By using qRT-PCR, western blot and luciferase assay, we identified SMAD4 as a direct miR-224 target mediating miR-224's effect on cell motility in CRC cells. We conclude that MiR-224 is associated with CRC malignancy, and functions as a metastasis activator by negative regulation of SMAD4.

Citation Format: HUI LING, George Calin, Milena Nicoloso, Mariko Ikuo. Biological and clinical significance of miR-224 in colorectal 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 1483. doi:10.1158/1538-7445.AM2014-1483
Abstract 1956: The basic helix-loop-helix (bHLH) transcriptional factor ATOH8 promotes the stemness of breast cancer cells via Oct4 and Nanog
Authors: Chang Antao , Chen Yanan , Shen Wenzhi , Gao Ruifang , Zhou Wei , Luo Yunping , Luo Na , Stupack Dwayne , Xiang Rong
Published: May 2015

Breast cancer is the leading cause of cancer mortality in women with a high incidence of recurrence and eventual treatment failure. The last decade has yielded substantial evidence that cancer stem cells (CSCs) contribute to tumor relapse, metastasis and chemoresistance. CSCs are regulated by complex interactions with the components of the tumor microenvironment through networks of cytokines and growth factors, including the IL6-STAT3 signaling pathway. Although the IL6 signaling pathway has been extensively characterized, the network of effector genes and proteins that regulates the CSC population remains poorly understood. Here, we show that ATOH8 is a downstream effector of IL6-STAT3 signaling which promotes the stemness of breast cancer cells. ATOH8 is significantly increased after IL6 stimulation and can be directly regulated by STAT3. Ectopic expression of ATOH8 in 4T1 or T-47D cells is sufficient to increases the CSC sub-population and enhances tumorogenesis in mice. Mechanistically, ATOH8 promotes the expression of Oct3/4 and Nanog, key regulators of CSC renewed. Accordingly, Knock-down of ATOH8 in EMT6 or MDA-MB-231 cells is associated with decreased expression of Oct3/4 and Nanog and reduces the CSC sub-population. In patients, ATOH8 expression is a negative prognostic indicator for overall survival. Together, the results identify ATOH8 as a downstream effector of IL6-STAT3 signaling that compromises long-term surviving in breast cancer.

Citation Format: Antao Chang, Yanan Chen, Wenzhi Shen, Ruifang Gao, Wei Zhou, Yunping Luo, Na Luo, Dwayne Stupack, Rong Xiang. The basic helix-loop-helix (bHLH) transcriptional factor ATOH8 promotes the stemness of breast cancer cells via Oct4 and Nanog. [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 1956. doi:10.1158/1538-7445.AM2014-1956
Abstract 2205: A three-gene signature predicts esophageal squamous cell carcinoma prognosis
Authors: Cao Huihui , Li Enmin , Xu Liyan
Published: May 2015

The 5-year survival rate of esophageal squamous cell carcinoma (ESCC) remains dismal despite improvements in treatments such as surgical resection and adjuvant chemoradiation, and current clinical staging approaches are limited in their ability to effectively stratify patients for treatment options. To improve the clinical risk assessment for ESCC patients, we developed an immunohistochemistry-based predictor model by evaluating the prognostic value of a three-gene signature, i.e. epidermal growth factor receptor, phosphorylated Specificity protein 1, and Fascin, which constitute a regulatory axis in ESCC cells as revealed by our previously study. The presence of this three-gene signature and the association with clinical outcomes were analyzed in 185 esophageal curative resection specimens (generation dataset), and the findings were validated using an independent cohort of 130 specimens (validation dataset). We revealed that the protein expression of this three-gene signature was highly predictive of ESCC survival in both generation and validation datasets (P=0.001). Regression analysis showed that this three-gene signature was an independently predictor of overall survival (hazard ratio=1.990 [95% CI, 1.256-3.154], P=0.003 in generation dataset; hazard ratio=2.358 [95% CI, 1.391-3.996], P=0.001 in validation dataset). Importantly, the predictive ability of the 3-gene signature was more robust than that of each individual factor. In conclusion, this immunohistochemistry-based molecular model accurately predicts ESCC patient survival, and thus could serve as a complement to current approached in clinical risk stratification.

Note: This abstract was not presented at the meeting.

Citation Format: Huihui Cao, Enmin Li, Liyan Xu. A three-gene signature predicts esophageal squamous cell carcinoma prognosis. [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 2205. doi:10.1158/1538-7445.AM2014-2205
Abstract 2293: A fast and simple method for whole-genome bisulfite library preparation from ultra-low DNA inputs
Authors: Giang Karolyn , Chung TzuHung , Sun Xueguang , Eden Marc E. Van , Jia Xi Yu
Published: May 2015

The distribution of 5-methylcytosine (5-mC) in DNA within the eukaryotic genome is known to greatly affect gene regulation and is currently a major topic of research. Studies on DNA methylation have been aided by advancements in bisulfite conversion and Next-Gen sequencing technologies which can provide single-base resolution of 5-mC across the entire genome. Many whole-genome bisulfite sequencing (WGBS) library preparation protocols designed for analysis of 5-mC distribution employ bisulfite to chemically convert unmethylated cytosine bases into uracil following the library preparation step. While these protocols produce reliable results, degradation of DNA is always an inherent issue when dealing with bisulfite conversion, and a large proportion of the adapterized library is often fragmented and can no longer be amplified. This requires high levels of DNA to serve as the input material. However, by rearranging the order of library preparation and bisulfite conversion, we developed a streamlined protocol that necessitates less input material for accurate whole-genome methylation analysis at single-base resolution. This unique workflow accommodates picogram quantities of starting material, making it ideal for analysis of precious samples that are of limited availability (e.g., FFPE). Comparison of sequencing data generated using this new library preparation method and data generated with established Reduced Representation Bisulfite Sequencing demonstrated a correlation of 0.95 in CpG sites with > 10X coverage in human DNA. Also, with only slight modification, this protocol is versatile enough to be used in the preparation of libraries for ChIP-Seq and RNA-Seq analyses.

Citation Format: Karolyn Giang, TzuHung Chung, Xueguang Sun, Marc E. Van Eden, Xi Yu Jia. A fast and simple method for whole-genome bisulfite library preparation from ultra-low DNA inputs. [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 2293. doi:10.1158/1538-7445.AM2014-2293
Abstract 239: Capsaicin may reduce the metastatic burden in the transgenic adenocarcioma of the mouse prostate (TRAMP) model
Authors: Venier Natalie A. , Yamamoto Toshihiro , Sugar Linda , Fleshner Neil , Klotz Laurence , Venkateswaran Vasundara
Published: May 2015

Introduction and Objective: A large body of evidence supports the role of dietary factors in prostate cancer development and progression. We are interested in investigating the chemopreventive potential of capsaicin, the active compound in chilli peppers that is traditionally used topically to treat various pain-related syndromes. Recently capsaicin has been demonstrated to have anti-carcinogenic properties in vitro through a number of different mechanisms. In our study we aim to study the chemopreventive properties of capsaicin using the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, a murine model that closely resembles the progression of human disease.

Methodology: Thirty-five six-week old TRAMP x C57Bk mice were randomized into two groups: control or capsaicin. Mice received either capsaicin (5 mg/kg body weight) or vehicle (saline) three times a week by oral gavage until the age of 30 weeks. Body weight was measured thrice weekly. All mice were sacrificed at 30 weeks. Body weight, genito-urinary (GU) weight, tumour burden were assessed. Prostate, seminal vesicles, lung, liver, esophagus, lymph nodes and pancreas were obtained for histological analysis. Serum was collected at the termination of the study for analysis of serum capsaicin concentration. Prostate tumours were analyzed by immunohistochemical analysis using proliferative and mechanistic markers. All tumours were scored by an on-site pathologist according to the histopathic grading scale described by Hurwitz AA, et al.

Results: Interim results revealed that higher percentage of high-grade cancer in control group (n = 18). The presence of PIN-like pre-cancerous lesions in only the treatment group and not the control group (n = 18). The capsaicin treated mice also demonstrated a reduced proportion of metastatic cancers compared to the control group. There were no significant changes in the GU wet weight between groups (n = 35). Immunohistochemical analysis of the prostate tumour is ongoing. Capsaicin was well tolerated, as there was no pathological liver or esophagus or gastrointestinal toxicities or difference in body weight between groups. Further results are currently under investigation.

Conclusion: Interim results suggest that oral administration of capsaicin is well tolerated and may reduce the metastatic burden in the TRAMP model. Ongoing studies are currently underway to delineate the mechanism of action.

Citation Format: Natalie A. Venier, Toshihiro Yamamoto, Linda Sugar, Neil Fleshner, Laurence Klotz, Vasundara Venkateswaran. Capsaicin may reduce the metastatic burden in the transgenic adenocarcioma of the mouse prostate (TRAMP) model. [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 239. doi:10.1158/1538-7445.AM2014-239
Abstract 2390: Identification of BRCA2 genetic interactors in an ES cell-based model
Authors: Ding Xia , Sharan Shyam K.
Published: May 2015

Mutations in breast cancer susceptible genes, BRCA1 and BRCA2 are the genetic factors conferring highest risk to develop breast cancer. Although BRCA proteins are known to maintain genomic stability mainly by homologous recombination-mediated DNA damage repair, detailed mechanisms of how BRCA loss induces tumorigenesis remain unclear. Interestingly, while inactivation of both alleles of BRCA1 or BRCA2 genes is necessary for tumor development, their loss in normal cells affects cell viability, indicating that cells lacking BRCA1 or 2 are able to survive and predisposed to tumorigenesis due to mutations in other genes such as those involved in cell cycle regulation or DNA damage response. In the current study, we identified some of the candidate genes using a mouse embryonic stem (mES) cell-based insertional mutagenesis screening. Overexpression of these genes rescued BRCA2 loss-induced lethality in mES cells. One candidate encodes a PDZ domain-containing protein, GIPC3. While overexpression of GIPC3 rescued BRCA2 loss efficiently, overexpression of its close homolog GIPC1 rescued much less efficiently. The GIPC3-rescued cells were hypersensitive to DNA damaging agents and exhibited an overall increase in genomic instability. Point mutation analysis showed several amino acids in the PDZ domain was critical for rescuing BRCA2 lethality, suggesting protein-protein interaction is responsible for the rescuing. We therefore performed IP-MS analysis and found that GIPC3 can interact with APPL1 and APPL2, two important mediators in AKT-mediated survival signal transduction pathway. We are now analyzing if these two molecules are potential down-stream factors of GIPC3 in rescuing BRCA2 loss-induced lethality. Meanwhile, we found that Gipc3 overxpression can partially rescue the lethality of Brca2ko/ko embryos. We are now also examining the expression of GIPC3 in human BRCA2-deficient tumors and we are monitoring tumorigenesis of GIPC3 transgenic mice in a K14-Cre;Brca2cko/cko mammary tumor model.

Citation Format: Xia Ding, Shyam K. Sharan. Identification of BRCA2 genetic interactors in an ES cell-based model. [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 2390. doi:10.1158/1538-7445.AM2014-2390
Abstract 2408: Identification of R-Spondin fusions in various types of human cancer
Authors: Cardona Gabriela Martinez , Bell Katherine , Portale Joseph , Gaffney Dana , Moy Christopher , Platero Suso , Lorenzi Matthew V. , Karkera Jayaprakash
Published: May 2015

Autocrine or paracrine constitutive Wnt pathway activation occurs at a high frequency in several tumor types. The R-spondin (RSPO) protein family is comprised of four secreted growth factors. The four paralogs share 40-60% pairwise amino acid sequence identity and are predicted to share substantial structural homology. RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands. A characteristic feature of all four RSPO members is their ability to activate β-catenin signaling and enhance WNT-mediated β-catenin activation. It has recently been described that recurrent gene fusions involving RSPO family members RSPO2 and RSPO3 occur in ∼10% of colon tumors.

In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these three (3) RSPO fusion transcripts in formalin-fixed paraffin embedded tissue (FFPET) samples. We examined 324 lung cancer, 81 colorectal cancer, 71 head & neck, 11 esophageal, 92 ovarian cancer, and 103 breast cancer FFPET samples for the presence of EIF3E(e1)-RSPO2(e1), PTPRK(e1)-RSPO3(e2), and PTPRK(e7)-RSPO3(e2). EIF3E(e1)-RSPO2(e1), a fusion which is expected to produce a functional RSPO2 protein driven by the EIF3E promoter, was identified in ∼1-2% of most of cancer types with the exception of breast cancer. The PTPRK(e1)-RSPO3(e2) fusion was expressed by ∼1-11% of the samples in the different cancers, making it the most prevalent of the fusions. PTPRK(e1)-RSPO3(e2) fusion is an in-frame fusion that preserves the entire coding sequence of RSPO3 and replaces its secretion signal sequence with that of PTPRK. The PTPRK(e7)-RSPO3(e2) fusion is also an in-frame fusion in which the RSPO3 native signal peptide is replaced by the secretion signal of PTPRK. The PTPRK(e7)-RSPO3(e2) was the least prevalent of all the fusions, positive samples were found exclusively in the head and neck (∼2%) and breast cancer samples (∼2%). All of the fusions detected were mutually exclusive. The RSPO gene fusions identified may provide new potential opportunities for therapeutic intervention.

Citation Format: Gabriela Martinez Cardona, Katherine Bell, Joseph Portale, Dana Gaffney, Christopher Moy, Suso Platero, Matthew V. Lorenzi, Jayaprakash Karkera. Identification of R-Spondin fusions in various types of human 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 2408. doi:10.1158/1538-7445.AM2014-2408
Abstract 2445: Estrogen promotes tumorigenesis by ADP-ribosyl-acceptor hydrolase 1 (ARH1)-deficient cells and mice
Authors: Kato Jiro , Bu Xiangning , Moss Joel
Published: May 2015

ADP-ribosylation, a post-translational modification in which the ADP-ribose of NAD is transferred to specific acceptors, is used by bacterial toxins to disrupt host cell biosynthetic, regulatory and metabolic pathways. Mammalian cells also use ADP-ribosylation reactions to regulate protein activity. The content of ADP-ribosylated acceptors is tightly regulated by enzymes that transfer ADP-ribose to acceptors i.e., ADP-ribosyltransferases (ARTs), and those that cleave the ADP-ribose acceptor bond, i.e., ADP-ribose-acceptor hydrolases (e.g., ARHs). ADP-ribosyl-acceptor hydrolase 1 (ARH1) is responsible for the hydrolysis of the α-ADP-ribosylarginine(protein) bond. We reported previously that ARH1-deficient mouse embryonic fibroblasts (MEFs) had greater proliferation rates, and formed more colonies in soft agar and tumors in nude mice than their wild-type (WT) counterparts. Consistent with these results with MEFs, ARH1-deficient (ARH1-/-) and heterozygous (ARH1+/-) mice developed tumors more frequently than their WT counterparts (Kato et al, 2011 Cancer Res.). Furthermore, the ARH1 gene from tumors isolated from ARH1+/- MEFs, and from tumors isolated from ARH1+/- mice contained mutations that affected function. Because loss of the functioning allele in ARH1+/- MEFs and mice resulted in tumorigenesis, we infer that ARH1 is a tumor suppressor gene.

Female ARH1-/- and ARH1+/- mice developed tumors and metastasis more frequently than male mice (e.g., adenocarcinomas, lymphomas), and developed tumors at a younger age than male mice. Consistent with the gender effect on incidence of tumors in ARH1-/- mice, tumor growth by ARH1-/- MEFs injected subcutaneously in an “estrogen group” (i.e., female, female ovariectomized [FO] supplemented with 17-β-estradiol [E2], male supplemented with E2) of nude mice was significantly faster than that seen in a “non-estrogen group” (i.e., male, FO) of mice. Pulmonary metastasis following intravenous injection of ARH1-/- MEFs in an “estrogen group” of nude mice were larger in size than those in a “non-estrogen group”. Further, estrogen promoted the survival of ARH1-/- MEFs in the circulation and thus enhanced metastatic potential. Our data demonstrate that control of protein-ADP-ribosylation by ARH1 is important for cell proliferation and tumorigenesis. Estrogen enhanced pulmonary metastasis, tumor growth and survival of circulating tumor cells.

Funding: Intramural Research Program, NIH, NHLBI.

Citation Format: Jiro Kato, Xiangning Bu, Joel Moss. Estrogen promotes tumorigenesis by ADP-ribosyl-acceptor hydrolase 1 (ARH1)-deficient cells and mice. [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 2445. doi:10.1158/1538-7445.AM2014-2445
Abstract 2477: Max inactivation in small cell lung cancer disrupts the MYC-SWI/SNF programs and is synthetic lethal with BRG1
Authors: Romero-Ferraro Octavio A. , Torres-Diz Manuel , Pros Eva , Moruno Antonio Gomez , Salama Sebastian Moran , Savola Suvi , Montuenga Luis M. , Pio Ruben , Yokota Jun , Sanchez-Cespedes Montse
Published: May 2015

Our knowledge of small cell lung cancer (SCLC) genetics is still very limited, amplification of L-MYC, N-MYC and C-MYC being some of the well established gene alterations. Here, we report our discovery of tumor-specific inactivation of the MYC-associated factor X gene, MAX, in SCLC. This is mutually exclusive with alterations at MYC and BRG1, the latter coding for an ATPase of the SWI/SNF complex. We demonstrate that BRG1 regulates the expression of MAX through direct recruitment to the MAX promoter, and that depletion of BRG1 strongly hinders cell growth, specifically in MAX-deficient cells, heralding a synthetic lethal interaction. Furthermore, MAX requires BRG1 to activate neuroendocrine transcriptional programs and to up-regulate MYC-targets, such as glycolytic-related genes. Finally, we observed genetic inactivation of the MAX dimerization protein, MGA, in lung cancers with wild type components of the SWI/SNF or MYC pathways. Our results provide evidence that an aberrant SWI/SNF-MYC network is essential for lung cancer development.

Citation Format: Octavio A. Romero-Ferraro, Manuel Torres-Diz, Eva Pros, Antonio Gomez Moruno, Sebastian Moran Salama, Suvi Savola, Luis M. Montuenga, Ruben Pio, Jun Yokota, Montse Sanchez-Cespedes. Max inactivation in small cell lung cancer disrupts the MYC-SWI/SNF programs and is synthetic lethal with BRG1. [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 2477. doi:10.1158/1538-7445.AM2014-2477
Abstract 2489: Glycoproteomic analysis of breast cancer cell lines for biomarker discovery
Authors: Arampatzidou Maria , Kullolli Majlinda , Pitteri Sharon J.
Published: May 2015

Glycosylation is one of the most ubiquitous protein post-translational modifications observed in eukaryotic organisms. In cancer, key cellular pathways and interactions, related to invasion and metastasis, are disrupted by altered protein glycosylation. Many current clinical cancer biomarkers are glycoproteins (i.e. CEA, CA15-5, CA125, and PSA). Glycosylation is known to be aberrant in breast cancer. However, systematic studies to identify glycoproteins in cancer have been limited, and typically focus on single proteins of interest or glycans released from proteins. We have developed a novel protein fractionation and enrichment strategy for glycoproteomic analysis of the secreted proteins, incorporating lectin affinity capture for glycoprotein enrichment, intact protein separation, and enzymatic digestion followed by LC-MS/MS analysis. Here, we apply this workflow to capture and analyze glycoproteins in the cancer secretome, which includes proteins secreted and/or shed from cancer cells into the extracellular space.

Four breast cancer cell lines (HCC70, HCC1806, BT474, and ZR75-1) and one normal mammary epithelial cell line (MCF10A) were subjected to glycoproteomic analysis. Proteins corresponding to 3360 unique gene names were identified. Subsequent data analysis yielded a variety of types of information about glycoproteins in the cell line secretomes. 1) Fucosylated and branched-sugar containing glycoforms of proteins were identified by lectin binding properties. For example, PHA-P/PHA-L lectins showed high affinity for aldolase, immunoglobulins, and inter-alpha-trypsin inhibitor, suggesting these proteins contain branched sugar structures. 2) Enrichment of some proteins was observed in breast cancer cell lines compared to the normal mammary epithelial cell line and vice versa. For example, high expression of fucosylated vimentin was observed in MCF10A, but not breast cancer cell lines. 3) Differences in glycosylation patterns of proteins were observed between cell lines. For example, the protease legumain was observed to be highly fucosylated in MCF10A, but had a higher affinity for PHA-P/PHA-L in HCC70. Additional substrates and inhibitors of legumain were also identified. Proteins of interest were further studied in additional follow-up experiments including western blotting and glycostaining. Additional proteins of interest were measured in plasma samples from women with (benign or malignant) breast lesions.

Citation Format: Maria Arampatzidou, Majlinda Kullolli, Sharon J. Pitteri. Glycoproteomic analysis of breast cancer cell lines for biomarker discovery. [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 2489. doi:10.1158/1538-7445.AM2014-2489
Abstract 3022: PAR-2 activation is required for cancer stem cells maintenance through upregulation of POSTN
Authors: Ma Yiming , Wang Hongying
Published: May 2015

Backgrounds: PARs are seven transmembrane-spanning domain G-protein coupled receptors (GPCRs) which act as targets of certain serine proteinases, such as thrombin and trypsin. PAR2 is the only member in the family which cannot be activated by thrombin. The studies in knockout mice revealed that PAR2 plays more important roles in tumor formation compared to other PARs.

Aims: To investigate the roles of PAR2 activation in cancer stem cell maintenance and possible mechanisms.

Methods and Results: In human colorectal cancer samples, PAR2 was significantly correlated with CD133 at mRNA level measured with real time PCR. With six colorectal cancer cell lines, it was found that the cell line with higher level of PAR2 expression presents higher rate of CD133+ cells and vice versa. To test whether PAR2 is required for cancer stem cell, we developed cell lines with knock down of PAR2 stablely. It was found that inhibition of PAR2 expression in cancer cells not only reduced the level of CD133 but also significantly blocked the ability of sphere formation in vitro. In addition, we found that PAR2 activation induced the expression of POSTN, which is a well established secreted protein related to cancer stem cell maintenance. Deficiency of PAR2 in cancer cell also reduced the level of POSTN. More importantly, recombinant POSTN protein was able to restore the ability of sphere formation decreased by PAR2 deficiency in cancer cells. Furthermore, Activation of PAR2 caused TGFβ activation without affecting levels of total TGFβ. Given that TGFβ(5ng/ml) only could stimulate POSTN expression, it is very possible that PAR2 stimulates POSTN expression through TGFβ-related pathway. Finally, to test the role of PAR2-induced POSTN in cancer stem cell maintenance in vivo, we measured the correlation of PAR2 and POSTN in paired primary and metastatic ovarian cancer samples. Not surprisingly, significant correlation of PAR2 and POSTN expression was only observed in metastatic ovarian cancer group, which express significant higher level of cancer stem cell-related genes compared with primary group.

Conclusion: Our study strongly indicated that PAR2 activation was involved in cancer stem cell maintenance through TGFβ- mediated POSTN expression.

Citation Format: Yiming Ma, Hongying Wang. PAR-2 activation is required for cancer stem cells maintenance through upregulation of POSTN. [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 3022. doi:10.1158/1538-7445.AM2014-3022
Abstract 3150: Exploring the role of Rap1Gap in the progression from DCIS to invasive breast carcinoma
Authors: Shah Seema , Osuala Kingsley , Mao Shihong , Li Quanwen , Sloane Bonnie , Krawetz Stephen , Mattingly Raymond R.
Published: May 2015

Understanding the underlying mechanisms that drive malignant progression of breast cancer is important. The purpose of this study is to define the role of Rap1Gap in the progression of ductal carcinoma in-situ (DCIS) to invasive cancer. We employed an in-vitro three-dimensional (3D) overlay model that provides a physiologically relevant microenvironment to study mechanisms of mammary gland development and malignant progression. We previously employed next generation sequencing (NGS) and identified 63 consistently upregulated genes in three DCIS lines (SUM102, SUM225, MCF10.DCIS) compared to non-transformed mammary epithelial cells. To gain insight into common transcriptional elements of co-regulated genes that may be critical to the DCIS phenotype, we have mined our NGS data using Frameworker (Genomatix). The 63 upregulated genes are associated with 244 candidate promoters out of a total of 82,703 promoters in the entire human genome. Our analysis shows that the common framework RXRF-ZF02-ZF02-PLAG-HDBP is only present in the promoters of three genes: RAP1GAP, SPRY4 and PDGFB. All of these genes are upregulated in our DCIS signature, which means that this framework is hugely enriched (336-fold) over what would be expected by chance. Rap1Gap has tumor suppressor properties; its functions, via regulation of Rap1 activity, include regulation of cell adhesion, suppression of cell proliferation and metastasis. In other epithelial cancers, loss of Rap1Gap has been linked to epithelial to mesenchymal transition (EMT) and invasion. To begin study of Rap1Gap in breast cancer progression, we performed immunohistochemistry. We show strong Rap1Gap staining in DCIS with little to no expression in myoepithelial and stromal components. Immunoblotting results show that Rap1Gap levels in MCF10.CA1d cells (a model of invasive carcinoma) are reduced compared to those in MCF10.DCIS cells. Rap1Gap expression is low in additional basal breast cancer cell lines, but high in luminal ER+ /PR+ cell lines. We propose that over-expression of Rap1Gap may serve as a tumor suppressive mechanism in DCIS; subsequent reduction of Rap1Gap may be a switch for EMT and progression to an invasive phenotype.

Citation Format: Seema Shah, Kingsley Osuala, Shihong Mao, Quanwen Li, Bonnie Sloane, Stephen Krawetz, Raymond R. Mattingly. Exploring the role of Rap1Gap in the progression from DCIS to invasive breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2014-3150