bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-103: Structural characterization of SPRY2-Cbl interactions
Authors: Zhang Na , Zhang Xuan , Washington Laurie , Anbanandam Asokan , Battaile Kevin , Gao Philip , Smalter Hall Aaron , Lovell Scott , Roy Anuradha , Hanzlik Robert , Perez Raymond
Published: August 2015

Sprouty 2 (SPRY2) is a feedback modulator of receptor tyrosine kinase (RTK)-ERK signaling, and a potential tumor suppressor. Cbl, an E3 ubiquitin ligase and scaffold protein, binds SPRY2 with high affinity, and sequesters or targets it for degradation. Cbl also binds several growth factor receptors. Hence SPRY2, Cbl and receptors are in a dynamic equilibrium. Truncation and site-mutagenesis studies previously mapped SPRY2-Cbl interactions to two discrete domains, TKB and RING. A pY binding pocket in the TKB SH2 region is critical for binding, but the role(s) of other TKB regions and/or RING are undefined. Interactions of three Cbl recombinant proteins (P47-G351 (TKBD), and two phosphomimetic RING active-conformation constructs, P47-D435 Y371D/E) with three SPRY2 peptides (P1: Q36-N53, a putative RING binder; P2: Q36-T60 (pY55), TKB binder; and P3: 54-60(pY55), a truncated TKB binder) were characterized by computational simulation, surface plasmon resonance spectroscopy (SPR), fluorescence polarization (FP), and X-ray crystallography. In silico modeling suggested possible low-affinity binding of P1 in the groove between TKBD SH2 and 4-helix (4H) regions and near the RING, but this was not confirmed by FP, SPR, or crystallography. Apo and P2 ligand-bound structures agreed with prior published data; three previously unknown 4H interaction sites were also identified. Data on the effects of site-specific 4H mutants on P2 binding affinities are pending. Binding affinities for P2 with the TKBD, Y371E, and Y371D constructs were 160±5, 92±9, and 45±3 nM, respectively, by SPR; FP results were similar. Direct interactions with the RING were not confirmed, but the higher P2 binding affinities observed with RING constructs suggest that this domain indirectly facilitates SPRY2 binding. Binding of P2 induced 16.9° rotation of the SH2 region and formation of new H-bonds to 4H, compressing the TKBD. Importantly, P4 induced identical conformational changes as P2, but had no direct contact with 4H. These observations are consistent with a refined model of SPRY2-Cbl interaction, where ligand docking at the pY SH2 site is sufficient to globally change TKB architecture, facilitating SPRY2 binding at previously unrecognized sites in the 4H region (Support: KU COBRE-PSF (NIH/RR017708, NIH/GM103420), KU Cancer Center, Kansas Bioscience Authority, and the George & Floriene Lieberman Endowment).

Citation Format: Na Zhang, Xuan Zhang, Laurie Washington, Asokan Anbanandam, Kevin Battaile, Philip Gao, Aaron Smalter Hall, Scott Lovell, Anuradha Roy, Robert Hanzlik, Raymond Perez. Structural characterization of SPRY2-Cbl interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-103. doi:10.1158/1538-7445.AM2015-LB-103
Abstract LB-115: Baseline neutrophil to lymphocyte ratio as a selection criterion for ipilimumab treatment in metastatic melanoma patients
Authors: Ferrucci Pier Francesco , Gandini Sara , Battaglia Angelo , Alfieri Salvatore , Pala Laura , Di Giacomo Annamaria , Amato Giovanni , Antonini Cappellini Gian Carlo , Giannarelli Diana , Cocorocchio Emilia , Martinoli Chiara
Published: August 2015

Background: Ipilimumab, a recently approved immunomodulatory drug, improves the survival of metastatic melanoma patients. Despite documented, durable objective responses, a significant number of patients fails to obtain clinical benefit from treatment. The aim of this study was to identify a criterion to select patients best suited to receive this drug.

Methods: Sixty-nine metastatic melanoma patients treated at the European Institute of Oncology with 3 mg/kg ipilimumab, were evaluated. Neutrophil to lymphocyte ratio (NLR) was calculated from pre-therapy full blood counts. Progression free survival (PFS) and overall survival (OS) were assessed using the Kaplan-Meier method, and multivariate Cox models were applied. Findings were independently validated on a cohort of 27 patients treated with 10 mg/kg ipilimumab at the University Hospital of Siena and on a cohort of 88 treated in Rome.

Results: Best overall response and disease control rates were 9% and 27%, respectively. Median PFS and OS were 3 and 5 months, respectively. Pre-therapy NLR was identified as the strongest and independent marker for treatment benefit in univariate and multivariate analyses. Patients with baseline NLR<5 had a significantly improved PFS (HR = 0.38; 95% CI: 0.22-0.66; P = 0.0006) and OS (HR = 0.24; 95% CI: 0.13-0.46; P<0.0001) compared with those with a NLR≥5.

Conclusions: Our findings show that baseline NLR is strongly and independently associated with outcome of patients treated with ipilimumab, and may serve as a selection criterion for this therapy.

Citation Format: Pier Francesco Ferrucci, Sara Gandini, Angelo Battaglia, Salvatore Alfieri, Laura Pala, Annamaria Di Giacomo, Giovanni Amato, Gian Carlo Antonini Cappellini, Diana Giannarelli, Emilia Cocorocchio, Chiara Martinoli. Baseline neutrophil to lymphocyte ratio as a selection criterion for ipilimumab treatment in metastatic melanoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2015-LB-115
Abstract LB-120: HER2 as a potential predictive marker and target for therapy in cervical cancer
Authors: Halle Mari K. , Ojesina Akinyemi I. , Tangen Ingvild L. , Holst Frederik , Engerud Hilde R. , Bertelsen Bjørn I. , Krakstad Camilla , Salvesen Helga B.
Published: August 2015

Cervical cancer is the third leading cause of cancer in the female population worldwide, causing the death of more than 240,000 patients annually in developing countries. Increased molecular knowledge is crucial to identify robust prognostic and predictive biomarkers that can better guide treatment. The tumor response to trastuzumab is well established and strongly links to HER2 expression status evaluated by the Hercep Test, hence, it is essential to define the level of expression of this receptor using the FDA-approved Hercep Test to stratify cervical cancer patients with potential benefits from trastuzumab treatment.

Comprehensive molecular characterization has been conducted on 88 paired normal and tumor cases identifying ERBB2 to be frequently altered in cervical cancers. We here explore the protein expression of HER2 by immunohistochemical staining in a larger validation series (n = 220) and relate HER2 expression to the ERBB2 gene alterations, patients molecular profile and clinicopathological features.

We find a highly significant correlation between Hercep Test score and mRNA ERBB2 expression (p<0.001). The level of ERBB2 mRNA was also significantly associated with copy number status (p = 0.007). Further clinocopathological parameters like high FIGO stage, high grade, adenocarcinomas and normal p53 status was significantly linked to high HER2 protein expression. Kaplan Meier survival analysis revealed that within the squamous cell carcinomas, high protein levels of HER2 was linked to poorer disease specific survival.

Our results show a link between ERBB2 amplification, high mRNA expression and protein levels for HER2 in aggressive cervical cancers. Further studies of HER2 as a potential predictive marker for response to trastuzumab treatment in cervical cancer are needed.

Citation Format: Mari K. Halle, Akinyemi I. Ojesina, Ingvild L. Tangen, Frederik Holst, Hilde R. Engerud, Bjørn I. Bertelsen, Camilla Krakstad, Helga B. Salvesen. HER2 as a potential predictive marker and target for therapy in cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-120. doi:10.1158/1538-7445.AM2015-LB-120
Abstract LB-135: E-cadherin as a buffer for mutated B-catenin: A genetic comparison of Apc and B-catenin mutations in vivo
Authors: Huels David J. , Ridgway Rachel A. , Sansom Owen J.
Published: August 2015

Wnt deregulation is a characteristic of many cancers, especially Colorectal Cancer (CRC). However, only the intestine harbours mutations in the tumour suppressor gene (TSG) Adenomatous Polyposis Coli (APC) which can be found in around ∼80% of CRC patients. Other cancers (e.g. hepatocellular carcinoma, solid pseudopapillary cancer) achieve Wnt deregulation by specific mutations of B-catenin (CTNNB1).

Using genetic approaches in the mouse intestine we compared mutations in APC and B-catenin in the level of Wnt activation, their kinetics and their ability to transform the intestinal epithelium in vitro and in vivo.

Deregulation of Wnt by mutation of B-catenin is much slower than by loss of APC, and only gradually increases Wnt activity. In contrast to loss of APC, Wnt deregulation by mutation of B-catenin is almost completely restricted to the small intestine of mice, and not the colon. We have evidence that the restriction of the B-catenin phenotype is due to different E-cadherin levels in the small intestine compared to the colon. Mutation of B-catenin in addition to reduction of E-cadherin levels synergises dramatically in vivo and is now able to transform the colon epithelium. We show that E-cadherin at the adherens junctions acts as buffer for the mutated B-catenin and slows down its accumulation in the cytoplasm.

These data shows a clear restriction of B-catenin mediated tumor initiation by E-cadherin. Interestingly, the other cancer types (hepatocellular carcinoma, solid pseudopapillary cancer) show a similar negative relationship of B-catenin mutation and E-cadherin expression.

Citation Format: David J. Huels, Rachel A. Ridgway, Owen J. Sansom. E-cadherin as a buffer for mutated B-catenin: A genetic comparison of Apc and B-catenin mutations in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-135. doi:10.1158/1538-7445.AM2015-LB-135
Abstract LB-147: Colonic crypt neuroendocrine cells regulate colonic stem cells during colorectal cancer development
Authors: Modarai Shirin R. , Opdenaker Lynn M. , Boman Bruce M.
Published: August 2015

Our studies on tissues from colon cancer patients show that in the development of CRC, stem cells (SC) overpopulation underlies tumor initiation and progression. Specifically, we have involved studying patients with Familial Adenomatous Polyposis (FAP) during CRC development, which carry cancer-predisposing germline mutations in the adenomatous polyposis coli (APC) gene. These results indicate that SC overpopulation caused by APC mutations is the driver mechanism. However, how the APC mutations cause SC overpopulation needs clarification.

Similar to SCs, Neuroendocrine cells (NECs) comprise ∼1% of the total cells in the colon and both reside in the crypt stem cell niche in the colonic epithelium. NECs are involved in autocrine and paracrine signaling to help regulate the function of surrounding cells. This leads to an overall goal of this project, to study the relationship between NECs and SCs in young-onset CRC, especially those individuals with a genetic predisposition to CRC. Our overall hypothesis is that NECs play a key role in maintaining the SC population in the normal colon and, aberrant signaling from NECs contributes to tumor initiation and increased stemness.

To begin to investigate underlying mechanisms, we studied human colon tissues with varying APC genotypes: (i) normal crypts (homozygous-wildtype APC); (ii) normal-appearing FAP crypts (heterozygous-mutant APC); (iii) adenomatous FAP crypts (homozygous-mutant APC); (iv) CRCs (homozygous-mutant APC& other mutations). Preliminary data from our laboratory has shown that in colon cancer progression: (i) there is a decrease in the number of cells that co-express both SC and NEC markers (ii) there is a decrease in the total number of NECs, and (iii) there is an increase in the total number of SCs. Furthermore, we have established an in vitro co-culture model system to study regulation of SCs by NECs. Results from our model show that SSTR1 and GLP-2R NEC signaling plays a role in modulating colonic SC populations. This in vitro model system can help analyze the interaction between SCs and NECs during CRC progression.

Taken together, our findings indicate that: (1) NE factors contribute to colonic SC regulation via an autocrine or paracrine mechanism in normal colon, and (2) dysregulation of these mechanisms contributes to colon cancer development. Using our established in vitro model system, we can track the changes in the SC and NEC population size, based on different conditions of anchorage independent growth and presence of potential therapeutic agents.

Citation Format: Shirin R. Modarai, Lynn M. Opdenaker, Bruce M. Boman. Colonic crypt neuroendocrine cells regulate colonic stem cells during colorectal cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-147. doi:10.1158/1538-7445.AM2015-LB-147
Abstract LB-150: Genetic depletion of DNMT1 reveals a DNMT1 threshold controlling DNA methylation in human cells
Authors: Cai Yi , Tsai Hsing-chen , Yen Ray-whay , Xia Limin , Zhang Yang , Baylin Stephen
Published: August 2015

Altered DNA methylation patterns are a central feature of most types of human cancer cells. However, the exact roles of DNA methyltransferases (DNMTs) in human DNA methylation maintenance are not well characterized. The long-established model that DNA methyltransferase DNMT1 maintains most DNA methylation in human cells has been challenged by multiple DNMT1 knockdown/knockout studies in the past decade. A potential role for de novo DNA methyltransferases DNMT3A and DNMT3B in maintaining DNA methylation in cancer cells has been suggested but is also not precisely known.

To investigate the above issues, we have utilized both genetic recombination and CRISPR technology to deplete DNMTs individually or in combination in human colorectal cancer cell line HCT116. Previously we created a strong HCT116 DNMT1 hypomorph (1KO) by genetic recombination. Although 1KO cells lost about 85% of DNMT1 protein, they still maintained the epigenetic silencing of tumor suppressor genes and showed only 20% decrease in overall DNA methylation. We now show that further depletion of DNMT1 in 1KO cells, to less than 1% of wild type HCT116 cells, resulted in elimination of most DNA methylation and reactivation of key tumor suppressor genes including p16. In addition, we found that DNA methylation in HCT116 cells is controlled by different DNMT1 thresholds. Surprisingly, when we disrupted DNMT3A or DNMT3B with CRISPR in HCT116 cells, we see altered DNA methylation patterns involving both gains and losses of DNA methylation. Since abnormal DNA hypermethylation in cancer often associates with epigenetic gene silencing, this observation is especially relevant for the function of DNMT3A mutations in acute myeloid leukemia. Finally, we demonstrated that depletion of DNMT1-targeting protein UHRF1 efficiently reduced DNA methylation in human colon cancer cells. Our results not only defined the distinct roles of individual DNMTs in human cancer cell DNA methylation maintenance but also suggested UHRF1 inhibition as an alternative approach to reverse abnormal DNA hypermethylation in human cancer cells.

Citation Format: Yi Cai, Hsing-chen Tsai, Ray-whay Yen, Limin Xia, Yang Zhang, Stephen Baylin. Genetic depletion of DNMT1 reveals a DNMT1 threshold controlling DNA methylation in human cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-150. doi:10.1158/1538-7445.AM2015-LB-150
Abstract LB-153: In vivo visualization of the targeted DNA methylation
Authors: Chen Yao-Li , Chen Shou-Tung , Chung Chun-Chun , Hsu Chia-Chen , Lin Ping-Yi , Chuang Wan-Ling , Chu Yi-Ju , Leu Yu-Wei , Hsiao Shu-Huei
Published: August 2015

DNA methylation is a dominant silencing epigenetic modification that is inheritable in somatic cells. The recruitment of DNA methylation was proven to be able to reshape the cell physiology and cause tumorigenesis. The reversible nature of the methylation modification also makes the DNA methylation a therapeutic target and the monitoring of the methylation/demethylation dynamics in vivo is thus important for the development of demethylation agents. A targeted DNA methylation method and a two-component reporter system were combined to monitor the increase and demolishment of DNA methylation in vivo. The two constructs of the reporter systems are: target gene promoter (like HIC1 and ENSA) linked with the Tet repressor gene; the other is the Tet operator linked with the reporter like EGFP (in cell), IFP and iRFP. Both constructs were co-transfected into cells like mesenchymal stem cells, MDA-MB-231 and HCT116, and stable clones with both constructs were isolated and validated. Targeted DNA methylation method was used to transfect the isolated cell and, as the increase of DNA methylation, the reporters expressed. The increased methylation was then quenched after the adding of demethylation agents. To test this system in vivo, cells with both constructs were injected into the immune-deficient mice and the tumors grew afterward. Targeted DNA methylation was performed in vivo and the increase of methylation/expression of reporter genes were observed by fluorescence-based tomography. This combined system is also effective to monitor the demethylation effects of the demethylation agents. (Supported by: NSC- 102-2320-B-194-003-MY3 and NSC-102-2314-B-371- 003-MY3, MOST Taiwan)

Citation Format: Yao-Li Chen, Shou-Tung Chen, Chun-Chun Chung, Chia-Chen Hsu, Ping-Yi Lin, Wan-Ling Chuang, Yi-Ju Chu, Yu-Wei Leu, Shu-Huei Hsiao. In vivo visualization of the targeted DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-153. doi:10.1158/1538-7445.AM2015-LB-153
Abstract LB-156: Increased Notch1 expression suppressed the growth but promoted the stemness of gastric cancer cells
Authors: Chiu Chia-Chen , Lo Chien-Ju , Shen Chien-Heng , Lu Chung-Kuang , Chou Jian-Liang , Chu Pei-Yi , Hsiao Shu-Huei , Tseng Min-Jen , Leu Yu-Wei , Hsu Chia-Chen
Published: August 2015

To explore if the Notch pathway were abnormally regulated during gastric tumorigenesis, abnormal DLL1 promoter methylation and inversely correlated mRNA expression were detected in cultured cell lines and patient samples. Elevated DLL1 expression was observed in gastric lesion like gastritis or erosion, but decreased DLL1 expression was detected in all four stages of gastric cancers (n = 44). To identify the effector genes and construct the possible Notch signaling network in gastric cancer, Myc-tagged Notch1 was overexpressed in gastric cancer cell line followed by the chromatin-immuno-precipitation sequencing (ChIP-seq) analysis. In ChIP-seq analysis, antibodies against Notch1, Rbpjκ, and Myc were used to identify the Notch1 targets while antibodies against trimethylated histone 3 at lysine 4 (H3K4me3) or lysine 27 (H3K27me3) were used to detect the associated epigenetic states. We found that the Notch1-overexpression suppressed the gastric tumoral growth, and the target loci-associated H3K4me3 (active chromatin) were reduced while the association with H3K27me3 (suppressive chromatin) were increased. Overall, the H3K4me3 and H3K27me3 combined bivalent marks were increased after Notch1-overexpression, and this was correlated with promoted stemness as indicated by the increased Oct4 and CD44 (reported gastric cancer stem cell like marker) expression. EZH2, a Polycomb protein, was found to be associated with Rbpjκ which further links the Notch signaling together with the maintenance of cell stemness. Therefore, Notch signaling might be critical for tumor formation in gastric cancer. (Supported by: NSC-102-2320-B-194-003-MY3 and NSC-102-2314-B-371-003-MY3, MOST Taiwan)

Citation Format: Chia-Chen Chiu, Chien-Ju Lo, Chien-Heng Shen, Chung-Kuang Lu, Jian-Liang Chou, Pei-Yi Chu, Shu-Huei Hsiao, Min-Jen Tseng, Yu-Wei Leu, Chia-Chen Hsu. Increased Notch1 expression suppressed the growth but promoted the stemness of gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-156. doi:10.1158/1538-7445.AM2015-LB-156
Abstract LB-162: Oxidative damage disrupts telomere integrity and leads to cell senescence
Authors: Tan Rong , Sun Luxi , Gao Ying , Lan Li
Published: August 2015

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising less than one percent of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence and cell death in U2OS, HeLa, and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including signal-free ends and sister-chromatid fusions, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.

Note: This abstract was not presented at the meeting.

Citation Format: Rong Tan, Luxi Sun, Ying Gao, Li Lan. Oxidative damage disrupts telomere integrity and leads to cell senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-162. doi:10.1158/1538-7445.AM2015-LB-162
Abstract LB-186: Fiber intake modulates the effect of alcohol on breast cancer
Authors: Romieu Isabelle
Published: August 2015

Alcohol intake has been related to an increase risk of breast cancer while dietary fiber intake has been inversely correlated to breast cancer risk. A beneficial effect of fiber on ethanol carcinogenesis through their impact on estrogen levels is still controversial.

The purpose of this study is to investigate the role of dietary fiber as potential modifying factors of the association of alcohol to breast cancer using data from the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

A prospective observational study of 334,850 women aged 35-70 years at baseline enrolled in the ten countries of the EPIC study and followed up for 11.0 years on average was conducted. Information on fiber and alcohol intake at baseline and average lifetime alcohol intake were calculated from country-specific dietary and lifestyle questionnaires. Hazard ratios (HR) and 95% confidence intervals (CI) of developing invasive breast cancer according to different levels of alcohol and fiber intake were calculated.

During 3,670,439 person-years, 11,576 incident breast cancer cases were diagnosed. Among women with low intake of fiber (<18.5 g/day), the risk of breast cancer per 10g/day of alcohol intake was 1.057 (1.032;1.082) while among women with high intake of fiber (>24.2 g/day) the risk of breast cancer was 1.012 (0.983;0.1.04) (Test for interaction p = 0.01). This modulating effect was stronger for fiber from vegetables and for estrogen positive tumors.

Our results suggest that fiber intake may modulate the positive association of alcohol intake to breast cancer risk. Women should be advised to control their alcohol intake, and increase their intake of fiber.

Citation Format: Isabelle Romieu. Fiber intake modulates the effect of alcohol on breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-186. doi:10.1158/1538-7445.AM2015-LB-186
Abstract LB-201: Functional role for tumor heterogeneity: Paracrine signaling between tumor subclones of mouse SCLC promotes metastasis
Authors: Kwon Min Chul , Proost Natalie , Sutherland Kate , Zevenhoven John , Berns Anton
Published: August 2015

Tumor heterogeneity might not only lead to pools of cells with a different resistance profile to therapy, the heterogeneity can also create a unique tumor-microenvironment. Earlier we have shown that clonal evolution in mouse SCLC can result in subclones with either neuro-endocrine (NE) and non-NE features. Co-grafting of such subclones endow the NE tumor cells with metastatic potential. To address the underlying mechanism we conducted a series of in vivo graft experiments and found that non-cell autonomous paracrine signaling is required in the subcutaneously grafted tumor to permit dissemination. The expression of transcription factor Pea3 in NE cells was identified as a functional downstream target of this paracrine interaction between the tumor subclones. Fgf2 secreted by non-NE subclones causes the expression of Pea3 in the NE cells through activation of Mapk pathway thereby temporarily potentiating its invasive capacity and facilitating intravasation into the circulation. These findings reveal a cooperating role of tumor cell subclones in mouse SCLC. Our data indicate that inhibition of the Fgf2/Mapk/Pea3 axis might be exploited to impair growth and metastatic spread of SCLC. Given the similarity in phenotypic tumor cell heterogenenity and signaling aberrations between mouse and human SCLC, inhibition of this signaling axis is therefore worth exploring in human SCLC.

Citation Format: Min Chul Kwon, Natalie Proost, Kate Sutherland, John Zevenhoven, Anton Berns. Functional role for tumor heterogeneity: Paracrine signaling between tumor subclones of mouse SCLC promotes metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-201. doi:10.1158/1538-7445.AM2015-LB-201
Abstract LB-229: Agonistic antibodies to costimulatory molecules, OX40 and GITR, significantly enhance the antitumor efficacy of Listeria monocytogenes (Lm-LLO)-based immunotherapy
Authors: Mkrtichyan Mikayel , Shrimali Rajeev , Ahmad Shamim , Abu Eid Rasha , Berrong Zuzana , Wallecha Anu , Petit Robert , Khleif Samir N.
Published: August 2015

Combinational anti-cancer immune therapies that target multiple tumor-mediated suppressive mechanisms or enhance effector immunity are emerging as promising strategies for cancer treatment. Recently, we demonstrated that the combination of Listeria monocytogenes (Lm-LLO)-based immunotherapy (ADXS11-001) and anti-PD-1 antibody results in improved anti-tumor therapeutic and immune efficacy. Interestingly, regardless of the presence of an antigen or treatment with anti-PD-1 antibody, treatment with the Lm-LLO alone significantly inhibited the suppressive arm of the immune system, including regulatory T cells and myeloid-derived suppressor cells. We therefore hypothesized that combination of ADXS11-001 with agonistic antibodies against co-stimulatory molecules will lead to further improvement of immune and therapeutic efficacy as a result of the combined down-regulation of the suppressive arm and the enhancement of the effector arm. Here we demonstrate that combining Lm-LLO-based immunotherapy with anti-OX40 or anti-GITR antibodies lead to significant inhibition of tumor growth and prolonged survival of animals. The complete regression of established TC-1 tumors occurs in 40% and 60% of animals treated with ADXS11-001 in combination with anti-OX40 and anti-GITR antibodies, respectively. We show that this therapeutic potency enhancement is due to a significant increase in antigen-specific immune responses along with ADXS11-001-mediated inhibition of suppressor cells. Thus, we believe that simultaneous stimulation of T cells while inhibiting the suppressor cells, specifically using Lm-LLO-based immunotherapy combination with agonistic anti-co-stimulatory molecule antibody is a feasible and translatable approach that can lead to overall enhancement of the efficacy of an anti-tumor immunotherapy.

Citation Format: Mikayel Mkrtichyan, Rajeev Shrimali, Shamim Ahmad, Rasha Abu Eid, Zuzana Berrong, Anu Wallecha, Robert Petit, Samir N. Khleif. Agonistic antibodies to costimulatory molecules, OX40 and GITR, significantly enhance the antitumor efficacy of Listeria monocytogenes (Lm-LLO)-based immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2015-LB-229
Abstract LB-251: Histone deacetylase inhibition for the treatment of epithelioid sarcoma; novel cross talk between epigenetic components
Authors: Lopez Gonzalo , Song Yechun , Ruder Dennis , Creighton Chad Creighton , Bolshakov Svetlana , Zhang Xiaoli , Lev Dina , Pollock Raphael
Published: August 2015

Objective

Enhanced knowledge of epithelioid sarcoma (ES) molecular determinants will hopefully aid the development of urgently needed targeted therapies. The hallmark loss of INI1 causes a disruption in epigenetics, potentially contributing to the development of the disease. Recently, we have demonstrated that HDAC inhibitors (HDACi) to have efficacious value for the treatment various genetically complex sarcomas. Here, we aim to evaluate the potential therapeutic efficacy of HDACi in ES.

Methods

Three ES cell lines were used for this study: Epi-544, HS-ES, VAESBJ. MTS and clonogenic assays were utilized to evaluate the effect of HDACi on cell line growth. Cell cycle FACS and annexin V PI/FACS analysis with WB for cleaved caspase 3 were used to determine the effects of HDACi on cell cycle and apoptosis, respectively. In vivo growth effects of HDACi were evaluated using SCID mouse ES xenograft models. An Illumina gene array was used to evaluate HDACi-induced gene change.

Results

HDACi inhibited ES cell growth and colony formation. HDACi induced G2 cell cycle arrest and enhanced apoptosis in all ES cell lines tested. Similarly, HDACi abrogated ES xenograft growth and increased apoptosis in vivo. Due to the HDACi mechanisms of function, one of the major

consequences of this therapy is gene expression modulation. Using an Illumina gene array, we sought to identify genes responsible for the in vitro and in vivo anti-ES effects observed. Array analysis showed Survivin be significantly down-regulated in response to HDACi treatment. HDACi-induced decrease of Survivin was confirmed via WB. For enrichment analysis of our genes set with published annotated gene signatures, the Molecular Signatures Database (MSigDB) was used. MSigDB analysis identified a curated gene set of regulated genes after EZH2 knockdown (KD) with significant commonality of our gene array. We demonstrated HDACi-induced decrease of EZH2. EZH2 KD resulted in reduced cell growth and increased apoptosis in VAESBJ cells.

Conclusion

HDACs play a role in the progression of ES, where HDACi abrogate ES cell growth and enhance apoptosis in vitro and in vivo. Gene array analysis revealed various genes modulated by HDACi that may contribute to the progression of the disease. Anti-ES effects may be in part through the HDACi-induced repression of EZH2.

Citation Format: Gonzalo Lopez, Yechun Song, Dennis Ruder, Chad Creighton Creighton, Svetlana Bolshakov, Xiaoli Zhang, Dina Lev, Raphael Pollock. Histone deacetylase inhibition for the treatment of epithelioid sarcoma; novel cross talk between epigenetic components. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-251. doi:10.1158/1538-7445.AM2015-LB-251
Abstract LB-257: Discovery and characterization of novel, highly potent and selective USP7 inhibitors
Authors: Gavory Gerald , O'dowd Colin , McClelland Keeva , Odrzywol Ewa , Brown Alan , Burton Stephanie , Barker Oliver , Burkamp Frank , Helm Matt , James Iain , Flasz Jakub , Arkoudis Elias , Harrison Tim
Published: August 2015

Over the past decade, protein ubiquitination has emerged as an important post-translational modification with roles in a plethora of cellular processes, and dysregulation in the ubiquitin proteasome system pathway (UPS) has been implicated in multiple human disorders including cancer.

Ubiquitin specific proteases (USPs) are cysteine proteases that catalyse the de-ubiquitination of numerous protein substrates including tumor suppressors and oncogenes, hence regulating their levels and/or functions. USPs therefore represent a fast growing and attractive target class for pharmacological intervention. USP7 in particular has attracted considerable attention for its implications in multiple key oncogenic pathways including most notably MDM2/p53, PTEN and DNA damage.

As part of a focussed effort towards targeting USPs, fragment screening was performed against a panel of family members, including USP7. Hits were identified by surface plasmon resonance and validated using orthogonal biophysical techniques (NMR, thermophoresis). Subsequent hit expansion identified molecules for which high-resolution co-crystal structures have been solved providing unique opportunities for structure-based design.

Medicinal chemistry optimisation has yielded a series of novel, reversible and potent USP7 inhibitors (e.g. IC50 < 10 nM) with excellent selectivity profiles against deubiquitinating (DUBs) and other non-related enzymes. These inhibitors are cell-permeable and also exhibit potent target engagement in cells (e.g. EC50 < 30 nM). In line with the mechanism of action, further cellular profiling has demonstrated effects on p53, p21 and MDM2 levels in a concentration-dependant manner. From a translational viewpoint, initial studies aimed at identifying cell lines sensitive to these inhibitors will also be discussed.

In summary, we report the discovery and detailed biochemical and cellular profiling of novel, potent and selective inhibitors of USP7. These molecules have drug-like properties and may provide opportunities for the development of new anticancer therapeutics.

Citation Format: Gerald Gavory, Colin O'dowd, Keeva McClelland, Ewa Odrzywol, Alan Brown, Stephanie Burton, Oliver Barker, Frank Burkamp, Matt Helm, Iain James, Jakub Flasz, Elias Arkoudis, Tim Harrison. Discovery and characterization of novel, highly potent and selective USP7 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2015-LB-257
Abstract LB-263: Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues
Authors: Skaer Chad W. , Pan Pan , Young Matthew , Wang Hsin Tzu , Oshima Kiyoko , Huang Yi-Wen , Stoner Gary D. , Lechner John , Wang Li-Shu
Published: August 2015

In our previous work, a diet containing 5% freeze-dried black raspberries (BRB) was shown to significantly inhibit colorectal cancer in ApcMin mice. Studies in recent years have shown that metabolomics may help to elucidate the anti-cancer effects of dietary factors. Initially, therefore, we evaluated the effects of black raspberries on the metabolomic profiles of C57BL/6J mice, the mouse strain from which ApcMin mice were derived. In this study, we report the effects of the BRB diet on the metabolomic profiles from C57BL/6J mice. After 8 weeks on a 5% BRB or control AIN-76A diet, feces, liver, and colon mucosa tissues from 9 BRB-fed mice and 7 control-diet fed mice were collected and profiled using mass spectrometry. A total of 424 named metabolites were observed in the liver, feces, and colon mucosa, with 115 metabolites whose levels were significantly different between BRB and control-diet mice (P<0.05). Of these 115 differentiated metabolites, 41 were altered in the colon mucosa, and 38 and 44 metabolites were different in the feces and liver, respectively. Almost all of the changed metabolites in the colon mucosa (36 of 41) were downregulated by the BRB diet, whereas in the feces and liver the majority were upregulated (58 of 82). Included in the upregulated metabolites were lipids and amino acids in the feces and liver tissue. Benzoate derivatives, the product of gut bacteria metabolism of BRB polyphenols, were also up in all tissues. Among the downregulated metabolites were lipids in the colon mucosa and lysolipids in the liver. The decreased levels of lipid metabolites, particularly fatty acids, in the colon mucosa of mice indicates that antagonizing fatty acid synthesis is a potential mechanism by which the BRB diet is altering energy utilization in the colon mucosa of mice.

Citation Format: Chad W. Skaer, Pan Pan, Matthew Young, Hsin Tzu Wang, Kiyoko Oshima, Yi-Wen Huang, Gary D. Stoner, John Lechner, Li-Shu Wang. Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2015-LB-263
Abstract LB-266: Suppression of colitis-associated colon carcinogenesis by the natural triterpenoid celastrol
Authors: Choi Sung Hee , Baker Emily , Kim Byung-Gyu , Tochtrop Gregory , Letterio John J.
Published: August 2015

Celastrol is a natural triterpenoid isolated from the medicinal herb Triptyrgium wilfordii, commonly known as the Thunder of God vine. Celastrol has been shown to have a broad range of anti-inflammatory and anti-cancer activities. Here we investigated whether celastrol administered as a dietary supplement can inhibit both colitis and colitis-associated colon cancer (CAC) in either genetic or chemically-induced CAC mouse models. As a genetic and spontaneous, inflammatory-driven intestinal neoplasia model, we generated a Smad4TKO / p27Kip1-/- (DKO) mouse in which a germ line p27Kip1 gene deletion (p27Kip1-/-) was combined with the T cell-restricted deletion of the Smad4 gene (Smad4TKO). All DKO mice developed spontaneous gastrointestinal inflammation and colon carcinoma by 3 months of age. Administration of celastrol as a dietary supplement (2 mg/day) to DKO mice increased survival and suppressed intestinal epithelial neoplasia by decreasing production of inflammatory mediators, including TNF-alpha, IL-6, IL-1beta and iNOS. We investigated the chemopreventive activity of celastrol in the chemically induced CAC mouse model using dextran sulfate sodium (DSS)/azoxymethane (AOM). Mice (C57/BL6, ages 6-8 weeks) received intraperitoneal injections with 10 mg/kg AOM, followed by exposure to 2% DSS in drinking water for 7 days. We repeated 3 cycles of 2% DSS, with 10 days of normal drinking water provided between cycles. Mice were either maintained on control diet or given celastrol (2 mg/day) diet beginning with first administration of DSS and continuing until termination of the experiment. Mice receiving celastrol diet showed less loss in body weight compared with the control diet group and marked protection from AOM/DSS-induced colon cancer. The incidence and number of colonic tumors in mice in the celastrol diet group were also significantly lower than those in control diet group. These results were accompanied by decreased expression of iNOS and lower production of pro-inflammatory cytokines. Our data demonstrate the protective effects of celastrol in the chemoprevention of CAC, was and this effect correlated with a dampening of iNOS expression and with a decrease of inflammatory cytokine production. These results highlight the potential of celastrol as an effective agent for the chemoprevention of CAC in humans.

Citation Format: Sung Hee Choi, Emily Baker, Byung-Gyu Kim, Gregory Tochtrop, John J. Letterio. Suppression of colitis-associated colon carcinogenesis by the natural triterpenoid celastrol. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2015-LB-266
Abstract LB-297: Integrative analysis identifies driver lncRNAs in prostate cancer
Authors: Guo Haiyang , Ahmed Musaddeque , Hua Junjie , Liang Yi , Langstein Jens , He Housheng Hansen
Published: August 2015

Genome-Wide Association Studies (GWAS) and whole-genome resequencing studies identified thousands of germline risk variants and somatic mutations in cancer. However, efforts to interpret these data have mainly focused on protein coding genes, despite the fact that majority of these genetic alterations locate outside of coding gene exons. A recent curation of over seven thousand RNA-seq data characterized more than fifty-eight thousand expressed long noncoding RNAs (lncRNAs) in human genome, twice as large as the number of protein coding genes. In this study, we integrated lncRNA gene expression, epigenetic and genetic alteration data to nominate potential driver lncRNAs in prostate cancer. We have identified eleven lncRNAs that are regulated by risk SNPs and somatic mutations through both cis and trans actions. The function of two of these lncRNAs in prostate cancer development and progression has been characterized. Our data suggests that lncRNAs may function as driving factors in prostate cancer development and progression.

Note: This abstract was not presented at the meeting.

Citation Format: Haiyang Guo, Musaddeque Ahmed, Junjie Hua, Yi Liang, Jens Langstein, Housheng Hansen He. Integrative analysis identifies driver lncRNAs in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-297. doi:10.1158/1538-7445.AM2015-LB-297
Abstract LB-301: MicroRNA target screen identifies NEK9 pathway as a crucial component regulating proliferation of cancer cells lacking functional p53
Authors: Tsuchiya Naoto
Published: August 2015

An abnormality of TP53 is one of the hallmarks of human cancer, and a major cause of human cancer development. Therefore, selective elimination of p53-inactivated cancer cells represents an ideal therapeutic strategy. We previously identified miR-22 as a tumor-suppressive miRNA, which induces apoptosis in p53 wild type (WT) cancer cells, but it causes cell cycle arrest in p53-deficient cancer cells (Cancer Res. 2011). This finding led us to hypothesize that miR-22 has a unique set of target genes that determined the fate of cancer cells according to p53 mutation status. In this study, using miR-22 as a screening tool, we tried to identify factors that play important roles in proliferation and cell cycle progression of p53-inactivated cancer cells. The screen identified NIMA-related kinase 9 (NEK9) as such a crucial regulator. Both in vitro and in vivo, depletion of NEK9 selectively inhibited cell proliferation of p53-inactivated cancer cells, and expression of the NEK9 open reading frame restored proliferation. The resultant cell cycle arrest occurred pre-dominantly in G1 phase and exhibited features of pre-mature senescence. Furthermore, repression of NEK9 affected expression of a wide range of genes encoding factors involved in mRNA processing, receptor signaling and cell cycle regulation, suggesting that NEK9 plays a role in controlling these processes in p53-deficient cells. Importantly, high level expression of both NEK9 and p53 mutant proteins in lung adenocarcinoma specimens were associated with poor prognosis, suggesting that NEK9 confers some advantages for malignant progression of p53 mutant cancer. Collectively, our findings indicated that a novel NEK9 network, which is specifically activated in p53-deficient context, is a crucial cellular component responsible for the growth capacity of cancer cells lacking functional p53.

Citation Format: Naoto Tsuchiya. MicroRNA target screen identifies NEK9 pathway as a crucial component regulating proliferation of cancer cells lacking functional p53. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-301. doi:10.1158/1538-7445.AM2015-LB-301