bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2442: EWS/FLI1 activates GLI1 indirectly through GLI2 in Ewing Sarcoma
Authors: Christensen Laura , May William A.
Published: July 2016

GLI1 expression in Ewing tumors has been shown to be enhanced by EWS/FLI1 expression in both model transformation systems and in Ewing cell lines. In Ewing cells, GLI1 has been shown to produce significant phenotypic effects consistent with a role in Ewing tumor formation and propagation. Also consistent with an important role is the demonstrated correspondence between the transcriptional target sets of EWS/FLI1 and of GLI1 in Ewing tumor cells. The principal physiologic developmental regulator of GLI1 is the diffusible Sonic Hedgehog ligand which binds the cell surface tumor suppressor Patched with consequent activation of the transmembrane protein Smoothened. In Ewing tumors, the enhancement of GLI1 expression has been shown to be Hedgehog independent and to occur downstream of Smoothened. Indeed, evidence has been published that EWS/FLI1 directly activates the GLI1 promoter. While there is experimental evidence that GLI1 inhibition may be clinically useful in Ewing tumors, preclinical evidence for one inhibitory approach has been disappointing. Interestingly, direct transcriptional activation of GLI1 has not been demonstrated to be of significance in other most other physiologic or pathologic contexts. We speculated that direct transcriptional activation of GLI1 by EWS/FLI1 might be only part of the story in Ewing tumors.

Since GLI2 is the most potent physiologic transcriptional activator of GLI1, we undertook to investigate the role of GLI2 in Hedgehog/GLI (HH-GLI) signaling in Ewing Tumors. We found that GLI2 is broadly expressed in Ewing tumors. Based on GLI2 overexpression and on shRNA knockdown of GLI2, we found that GLI2 expression increases GLI1 expression in Ewing cell lines. Via GLI2 knockdown, we have found that GLI2 has biologic effects similar to or greater than GLI1 in Ewing cells. Finally, we demonstrate that EWS/FLI1 enhances nuclear localization of GLI2, suggesting an additional indirect mechanism by which EWS/FLI1 activates HH-GLI signaling and GLI1 expression in Ewing tumors.

These data indicate that GLI2 is an active player in HH-GLI pathway activation in Ewing tumors. They also suggest that measures which target GLI2 may have equal or greater efficacy to those aimed at GLI1.

Citation Format: Laura Christensen, William A. May. EWS/FLI1 activates GLI1 indirectly through GLI2 in Ewing Sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2442.
Abstract 2589: Dissecting extrinsic and intrinsic cancer risk
Authors: Wu Song , Powers Scott , Zhu Wei , Hannun Yusuf
Published: July 2016

Cancers were once thought to originate from mature tissue cells that underwent dedifferentiation in response to cancer progression. Today, cancers are proposed to originate from the malignant transformation of normal tissue progenitor and stem cells, although this is not uniformly accepted. Nevertheless, recent research has highlighted a strong correlation between tissue-specific cancer risk and the lifetime population size and cumulative number of cell divisions of tissue-specific stem cells. However, there has been extensive controversy regarding the conclusion that this correlation implies that there is a very high unavoidable risk for many specific cancers that are due solely to the intrinsic baseline population size of tissue-specific stem cells. This issue has become a key public health debate with dissemination of the ‘bad luck’ hypothesis. Here we provide strong evidence that unavoidable intrinsic risk factors contribute only modestly (<10-30%) to the development of many common cancer types. In an initial approach, we demonstrate that the correlation between stem-cell division and cancer risk does not distinguish between the effects of intrinsic and extrinsic factors. We then develop a novel data-driven approach to estimate intrinsic risk by considering the lower bound risk value for most cancers as a limiting condition for contribution of intrinsic processes. Furthermore, we present an independent mathematical model that capitalizes on the accumulation of endogenous mutations in stem cells. Using mutation rates as a starting point, we found that rates of intrinsic mutations are not sufficient to account for the observed lifetime cancer risk, even in the conservative estimate that all these mutations are due to intrinsic processes alone. Our results are consistent with vast epidemiological data at population level and also mutational signature data at molecular level. The consensus suggests that cancer risk is likely to be heavily influenced by extrinsic factors, and therefore is possibly modifiable and preventable. These results carry immense consequences for strategizing cancer prevention, research, and public health. [Note: this work is in press in Nature.]

Citation Format: Song Wu, Scott Powers, Wei Zhu, Yusuf Hannun. Dissecting extrinsic and intrinsic cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2589.
Abstract 259: Modulation of doxorubicin actions on HCC cells by insulin-like growth factor-I
Authors: Carr Brian I. , Refolo Maria Grazia , D’Alessandro Rosalba , Carella Nicola , Messa Caterina , Cavallini Aldo
Published: July 2016

Tumor microenvironment is increasingly understood to influence tumor biology in many tumor types, including HCC. We recently showed that platelets could modulate Doxorubicin (Dox)-mediated HCC growth inhibition (Refolo MG et al. Modulation of doxorubicin mediated growth inhibition of hepatocellular carcinoma cells by platelet lysates. Anticancer Agents Med Chem. 2014;14:1154-60). Platelets contain cytokines and growth factors, including IGF-1, PDGF, FGF, EGF and serotonin. We found that Insulin-like growth factor-1 (IGF-1) shifted the Dox growth-inhibitory dose-response in the range of 0.1-1.0 μM on Hep3B, HepG2, PLC/PRF/5 human HCC lines, all of which became less sensitive to both Dox-mediated inhibition of growth and apoptosis. Similar antagonism by IGF-1 was found for Dox-mediated inhibition of cell migration (scratch assay) and invasion (Boyden chamber). This antagonism of Dox effects on cell growth, migration and apoptosis by IGF1, was blocked by GSK1838705A, an IGF-1R inhibitor. Furthermore, when platelet extracts were used to antagonize Dox inhibition of growth and migration, the platelet effects were also blocked by addition of GSK1838705A to the cultures, showing that the platelet effects were largely mediated by IGF-1. Dox caused a decrease in the levels of anti-apoptosis markers Bcl-2, Bcl-xL and survivin as seen on WB, whereas levels of all three anti-apoptosis markers were increased by IGF-1 action. These actions of IGF-1 were also blocked by GSK 1838705A. Furthermore, Dox caused a decrease in the levels of P-ERK, P-p38 and P-STAT3 (ser727). These signaling molecule decreases were also reversed by the presence of IGF-1.

Conclusions. Dox actions on HCC cells can be antagonized by platelets and specifically by platelet-associated IGF-1. IGF-1 antagonists appear to be attractive enhancers of sensitivity to Dox actions.

Citation Format: Brian I. Carr, Maria Grazia Refolo, Rosalba D’Alessandro, Nicola Carella, Caterina Messa, Aldo Cavallini. Modulation of doxorubicin actions on HCC cells by insulin-like growth factor-I. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 259.
Abstract 2595: Texture variation on a mammogram and risk of breast cancer
Authors: Rice Megan , Bertrand Kimberly , Heine John , Rosner Bernard , Tamimi Rulla
Published: July 2016

Mammographic density is one of the strongest risk factors for breast cancer. The most widely used measure of mammographic density is based on the percentage of dense tissue on a mammogram (percent mammographic density). However, there is additional information in mammographic images not captured by current mammographic density measurements including heterogeneity in patterns of breast density, often referred to as ‘texture’. More recent research suggests that textural features on a mammogram are associated with breast cancer risk independent of percent mammographic density. Therefore, we evaluated an automated measure called V, which measures the grayscale variation within a mammogram, in relation to subsequent breast cancer risk in 834 breast cancer cases (426 premenopausal and 408 postmenopausal) and 1805 controls (978 premenopausal and 827 postmenopausal) from a nested case-control study of breast cancer in the Nurses’ Health Studies (NHS). Among all women, V was modestly correlated with percent mammographic density (r = 0.48, p<0.01); this correlation did not vary substantially by menopausal status. Among premenopausal women, those in the highest quartile of V had a significantly higher risk of breast cancer (OR = 1.84, 95%CI: 1.30-2.61) compared to those in the lowest quartile. This association was similar, though more modest, among postmenopausal women (OR = 1.54, 95%CI: 1.08-2.19). These associations somewhat attenuated after adjustment for percent mammographic density in both premenopausal women (OR comparing extreme quartiles = 1.41, 95%CI: 0.97-2.03) and in postmenopausal women (OR comparing extreme quartiles = 1.21, 95%CI: 0.81-1.81). Future work will examine the associations between V and risk of breast cancer subtypes (e.g., by ER/PR tumor expression) as well as examine the associations between other measures of texture including markovian, run-length, laws, wavelet, fourier, and power features with breast cancer risk.

Citation Format: Megan Rice, Kimberly Bertrand, John Heine, Bernard Rosner, Rulla Tamimi. Texture variation on a mammogram and risk of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2595.
Abstract 2608: Dietary polyunsaturated fatty acids modulate adipose inflammation and the adipokine secretome to influence mammary stem cell self-renewal
Authors: Esper Raymond M. , Hill Evan , Jiang Yan , Aslam Nadeem , Simon Becky , Wicha Max , Smith William , Brenner Dean E.
Published: July 2016

Obesity strongly increases risk for multiple malignancies. One potential contributor may be the chronic low-grade inflammation that is likely driven by dysfunctional adipose characterized by aberrant adipokine production and macrophage activation/polarization. In adipose tissue, free fatty acids and their lipid mediators serve as important regulators of adipokine gene transcription. The adipocyte fatty acid composition is influenced by dietary availability. Here we present data demonstrating that dietary fish oil supplementation in obese rats (achieving a 2.5:1 ω6:ω3 PUFA ratio, comparable to a Mediterranean diet) attenuates adipose inflammation characterized reduction in the M1/M2 macrophage polarization ratio compared to a typical Western diet with a 20:1 ω6:ω3 PUFA ratio. We developed a novel adipose explant culture system to directly assay the secretome, and demonstrate that quenching adipose inflammation in obese animals is associated with a favorably rebalanced adipokine milieu, most notably increased adiponectin concentration and decreased leptin secretion. We previously showed that these two adipokines differentially modulate mammary stem cell self-renewal; here, we studied the effect of adipose conditioned media from obese rats on mammary stem cells using the mammosphere formation assay. Adipokines from obese rats fed the high-ω6 diet increased mammary stem cell symmetric self-renewal by 86.7 ± 7.5% (± SEM), whereas adipose-derived factors from the obese animals fed a low-ω6 diet supplemented with fish oil reduced the number of secondary mammospheres by 30.3 ± 10.1% by promoting either apoptosis, quiescence, or symmetric division-differentiation of the primary stem cells. Using a neutralizing antibody and a soluble receptor, we demonstrate that leptin contributes only 19% of the adipose-derived effect on mammary stem cell self-renewal. Taken together, our data suggests that a simple dietary intervention to modify the dietary PUFA ratio is an effective method to quench adipose inflammation and rebalance the adipokine milieu, which may modulate the mammary stem cell pool. If normal stem cells can give rise to breast cancer as has been theorized, then dietary interventions to quench adipose inflammation and reduce the rate of symmetric stem cell self-renewal might protect against the increased incidence of breast cancer seen in obese post-menopausal women.

Citation Format: Raymond M. Esper, Evan Hill, Yan Jiang, Nadeem Aslam, Becky Simon, Max Wicha, William Smith, Dean E. Brenner. Dietary polyunsaturated fatty acids modulate adipose inflammation and the adipokine secretome to influence mammary stem cell self-renewal. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2608.
Abstract 2609: Epigenetic reactivation of TIMP-3 in human prostate cancer cells by green tea polyphenols
Authors: Deb Gauri , Thakur Vijay S. , Shankar Eswar , Gupta Sanjay
Published: July 2016

Metastasis is responsible for more than 90% of prostate cancer-associated mortality in the United States. Metastasis is facilitated by remodeling of the extracellular matrix by a family of proteolytic enzymes known as matrix metalloproteinases (MMPs). These zinc-dependent proteases elicit pivotal role in the proteolytic degradation of structural components of extracellular matrix contributing to the metastatic process. High levels of MMP-2/9 in the plasma and urine has been correlated with metastasis. MMPs are produced in a latent form (pro-MMP) that require activation and are inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). TIMPs inhibit MMPs by binding in a 1:1 stoichiometry through strong noncovalent interactions. In prostate cancer, low levels of TIMP3 has been associated with aggressive tumor phenotype and poor disease-free survival. Loss of TIMP3 in prostate cancer is attributed to epigenetic silencing tipping the balance towards active MMPs driving metastasis. Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) has been shown to suppress prostate cancer metastasis, however the mechanism has not been fully elucidated. We determined whether treatment with GTP/EGCG has ability to restore induction of TIMP3 and play a key role in suppressing invasiveness in prostate cancer. Treatment of prostate cancer LNCaP and DUPRO cells with 10μg/mL GTP and 20μM EGCG for 72 h significantly induced TIMP3 mRNA and protein levels. Silencing of enhancer of zeste homolog 2 (EZH2) and class I histone deacetylases (HDACs) significantly increased the expression of TIMP3 which was independent of DNA hypermethylation. Treatment of prostate cancer cells with GTP/EGCG significantly reduced EZH2 and class I HDAC protein levels; H3K27 trimethylation activity; and transcriptional activation of TIMP-3 was found to be associated with decreased EZH2 localization and H3K27 trimethylation enrichment at the TIMP3 promoter with a concomitant increase in histone H3K9/18 acetylation. EGCG/GTP treatment also caused a global reduction in histone methylation thereby increasing histone acetylation to revive and activate TIMP3 levels. Furthermore, EGCG/GTP exposure significantly reduced MMP2/9 gelatinolytic activity and abrogated invasive and migration capabilities in prostate cancer cells. Taken together, our findings suggests that TIMP-3 induction could be a key epigenetic event modulated by GTPs in restoring MMP: TIMP balance to delay prostate cancer invasion and metastasis.

Citation Format: Gauri Deb, Vijay S. Thakur, Eswar Shankar, Sanjay Gupta. Epigenetic reactivation of TIMP-3 in human prostate cancer cells by green tea polyphenols. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2609.
Abstract 2660: A renal CpG island methylator phenotype (R-CIMP) in kidney tumors associated with germline mutations of FH and SDHB
Authors: Ricketts Christopher , Killian J. Keith , Vocke Cathy D. , Sourbier Carole , Yang Youfeng , Merino Maria J. , Meltzer Paul S. , Linehan. W. Marston
Published: July 2016

Germline mutations of the genes encoding the Krebs cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDHB, SDHC, SDHD) have both been associated with an increased risk of renal cell carcinoma (RCC). These renal tumors demonstrate complete loss of enzyme activity and significantly increased levels of intracellular fumarate or succinate that are predicted to infer with the actions of multiple cellular processes primarily through the inhibition of 2-oxoglutarate-dependent dioxygenases, such as the prolyl hydroxylases that regulate the HIF pathway and the TET enzymes that regulate DNA methylation.

This study evaluated and compared the genome-wide methylation profiles of 33 tumor samples (including 9 FH -/- and 7 SDHB -/- tumors), 10 associated normal samples and 13 cell-lines (including cell-line models derived from FH -/- and SDHB -/- tumors) using the Illumina HumanMethylation450 BeadChip arrays. Tumors derived from patients

with germline FH or SDHB mutations demonstrated a distinct pattern of hypermethylation across the genome with enrichment for hypermethylation within the CpG island regions. SDHB mutation associated tumors demonstrated that was more variable and less extensive pattern of hypermethylation than the tumors associated with germline FH mutations. Cell-line models derived from HLRCC or SDHB tumors demonstrated patterns of hypermethylation similar to the patterns observed in the tumors, yet re-expression of either wild-type FH or SDHB within these cell-line models did not alter the methylation profiles. All the HLRCC and SDHB-RCC cell lines, including the cell-line re-expressing of either wild-type FH or SDHB, demonstrated growth inhibition and reduced invasion rates in response to treatment with 5-aza-2′-deoxycytidine.

Thus, renal cell carcinomas associated with FH or SDHB germline mutation demonstrate a specific R-CIMP hypermethylation profile that differentiates these tumors from tumors derived from other RCC susceptibility syndromes. These tumors represent the most aggressive

types of familial-associated RCC and this could provide a useful biomarker for identifying these tumors and de-methylating agents, such as 5-aza-2′-deoxycytidine, may be effective as part of a therapeutic regime for the treatment of these tumors.

Citation Format: Christopher Ricketts, J. Keith Killian, Cathy D. Vocke, Carole Sourbier, Youfeng Yang, Maria J. Merino, Paul S. Meltzer, W. Marston Linehan. A renal CpG island methylator phenotype (R-CIMP) in kidney tumors associated with germline mutations of FH and SDHB. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2660.
Abstract 2667: Platelet microparticles infiltrating solid tumors transfer miRNAs and modulate tumor angiogenesis and growth
Authors: Michael James V. , Wurtzel Jeremy G.T. , Mao Guang Fen , Rao A. Koneti , Hoffman Nicholas E. , Rajan Sudarsan , Madesh Muniswamy , Jana Fabian , Nieman Marvin , Rowley Jesse W. , Weyrich Andrew S. , Goldfinger Lawrence E.
Published: July 2016

Platelet-derived microparticles are associated with enhancement of metastasis and poor cancer outcomes. Platelet microparticles can transfer platelet microRNAs (miRNAs) to vascular cells upon co-incubation in vitro, but the contributions of platelet microparticles and miRNAs to tumor progression are still poorly understood. Tumor vasculature is highly permeable, allowing the possibility of platelet microparticle-tumor cell interaction in primary solid tumors. Here we show that platelet microparticles infiltrate solid tumors in humans and mice, attach to tumor cells, and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo as well as in vitro. MiR-24 was a major species in this transfer. We identified RNA targets of platelet-derived miR-24 in lung carcinoma cells, which included mt-Nd2, a mitochondrial mRNA which lacks a 3’-UTR, and Snora75, a non-coding small nucleolar RNA. These RNAs were depleted in platelet microparticle-treated tumor cells, resulting in mitochondrial dysfunction and tumor cell growth inhibition, in a miR-24-dependent manner. Blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by platelet microparticle transfusion. Thus, platelet microparticles inhibit lung carcinoma cell proliferation and solid tumor growth via transfer of miR-24. However, global depletion of platelet miRNAs by platelet-specific deletion of Dicer1 inhibited tumor angiogenesis, platelet microparticle infiltration, and delayed tumor growth in mice. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via microparticles, regulate tumor cell gene expression, and modulate tumor progression, whereas platelet miRNAs promote tumor angiogenesis. These findings shed novel insight onto mechanisms of horizontal gene transfer and add multiple layers to the regulatory roles of miRNAs and platelet microparticles in tumor progression.

Citation Format: James V. Michael, Jeremy G.T. Wurtzel, Guang Fen Mao, A. Koneti Rao, Nicholas E. Hoffman, Sudarsan Rajan, Muniswamy Madesh, Fabian Jana, Marvin Nieman, Jesse W. Rowley, Andrew S. Weyrich, Lawrence E. Goldfinger. Platelet microparticles infiltrating solid tumors transfer miRNAs and modulate tumor angiogenesis and growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2667.
Abstract 2679: Induction of β-globin protects circulating tumor cells from oxidative stress during dissemination
Authors: Zheng Yu , Miyamoto David T. , Wittner Ben S. , Sullivan James P. , Aceto Nicola , Vincent Jordan Nicole , Yu Min , Karabacak Nezihi Murat , Comaills Valentine , Morris Robert , Desai Rushil , Desai Niyati , Emmons Erin , Lee Richard J. , Wu Chin-Lee , Sequist Lecia V. , Haas Wilhelm , Ting David T. , Toner Mehmet , Ramaswamy Sridhar , Maheswaran Shyamala , Haber Daniel A.
Published: July 2016

Identification of candidate metastasis genes has traditionally resulted from comparison of primary and metastatic tumor specimens. However, Circulating Tumor Cells (CTCs) contain metastatic precursors that are present transiently in the bloodstream and their analysis may reveal additional pathways that are induced for a limited time, as they invade and survive within the vasculature. By comparing transcriptome profiles of CTCs from breast, prostate and lung cancers with their primary tumor of origin, we observed consistent and significant induction of the β-globin gene (HBB) within CTCs. In contrast, expression of α-globin, its binding partner within hematopoietic cells, is not coordinately upregulated. The tumor-specific origin of HBB was further confirmed by analysis of human xenografts-derived CTCs in mice, where human-specific HBB polymorphisms are readily distinguishable in the murine background. In cultured cancer cells, we further demonstrate that induction of HBB is triggered by exposure to reactive oxygen species (ROS), and we identify KLF-family transcriptional regulators that mediate this effect. To investigate the function of aberrant β-globin expression within CTCs, we performed shRNA-mediated knockdown of HBB in breast CTC-derived cultures. Cells with depleted HBB expression displayed elevated intracellular ROS levels, increased sensitivity to hydrogen peroxide, and impaired metastatic potential in mouse models. Taken together, these observations suggest that β-globin, a component of functional hemoglobin in red blood cells, is deregulated in disseminated tumor cells, where it may function as a ROS scavenger, reducing oxidative stress and facilitating cancer metastasis.

Citation Format: Yu Zheng, David T. Miyamoto, Ben S. Wittner, James P. Sullivan, Nicola Aceto, Nicole Vincent Jordan, Min Yu, Nezihi Murat Karabacak, Valentine Comaills, Robert Morris, Rushil Desai, Niyati Desai, Erin Emmons, Richard J. Lee, Chin-Lee Wu, Lecia V. Sequist, Wilhelm Haas, David T. Ting, Mehmet Toner, Sridhar Ramaswamy, Shyamala Maheswaran, Daniel A. Haber. Induction of β-globin protects circulating tumor cells from oxidative stress during dissemination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2679.
Abstract 3323: Breast cancer stem cell culture and enrichment using poly(ϵ-caprolactone) 3D scaffolds
Authors: Rabionet Marc , Palomeras Sonia , Ferrer Ines , Sarrats Ariadna , Giro-Perafita Ariadna , Garcia-Romeu Maria Luisa , Ciurana Joaquim , Puig Teresa
Published: July 2016

Introduction: Cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies and a tendency for post-treatment recurrence. Increasing knowledge about CSCs phenotype and functions is needed to investigate new therapeutic strategies against CSC population. Poly(ϵ-CaproLactone) (PCL) as biocompatible and free toxic dye polymer, have been used to manufacture scaffolds, solid structures suitable for 3D cancer cell culture. It has been described that scaffold cell culture enhances the CSCs population.

Experimental procedures: A RepRap BCN3D+ printer and 3.5 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several product features of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures.

Summary of the new, unpublished data: Results shown that the 60º scaffolds were more suitable for 3D culture than the 45º and 90º ones. Moreover, 3D culture experiments both, in adherent and non-adherent conditions, showed a significant increase on MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and to enrich the CSCs population. Basically cells population growth with a 3D culture better and higher in value as it is demonstrated within the work.

Conclusions: Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and to enrich the CSCs population for further pre-clinical studies on CSC molecular characterization and treatment.

Citation Format: Marc Rabionet, Sonia Palomeras, Ines Ferrer, Ariadna Sarrats, Ariadna Giro-Perafita, Maria Luisa Garcia-Romeu, Joaquim Ciurana, Teresa Puig. Breast cancer stem cell culture and enrichment using poly(ϵ-caprolactone) 3D scaffolds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3323.
Abstract 3377: Bevacizumab-improved distribution of paclitaxel in ovarian cancer xenografts potentiates antitumor efficacy
Authors: Cesca Marta , Morosi Lavinia , Berndt Alexander , Fuso Nerini Ilaria , Decio Alessandra , Zucchetti Massimo , Giavazzi Raffaella
Published: July 2016

Angiogenesis inhibitors have shown antitumor efficacy when combined with chemotherapy. It has been proposed that this potentiation is related to better drug delivery to the tumor due to improved vessel functionality. This work combined classical pharmacokinetics and imaging analysis to study the delivery and distribution of paclitaxel after bevacizumab in ovarian cancer.

The study was conducted on ovarian cancer models (A2780-1A9 and IGROV-1) xenografted orthotopically under the bursa of the nude mouse ovary. Mice bearing tumors in the ovary, treated with bevacizumab (150μg/mouse) and 24 hours later with paclitaxel (60mg/kg), were sacrificed and tumor excised for analysis, or monitored for anti-tumor activity. Paclitaxel spatial distribution after bevacizumab was analyzed by MALDI-Mass Spectrometry Imaging (MALDI-MSI) on frozen tumor slices, and compared with adjacent histochemical images stained for CD31 (vessel analysis) and Ki-67 (proliferation/necrosis); total paclitaxel in tumor homogenates, liver and plasma was evaluated by high-performance liquid chromatography (HPLC). Tumor growth of A2780-1A9 and IGROV-1, carrying the firefly luciferase gene luc2, was monitored by Bioluminescence imaging (BLI) and survival recorded.

Bevacizumab decreased tumor vessel number/size compared to vehicle treated mice, but potentiated the antitumor activity of paclitaxel in both models. MALDI-MSI showed that after bevacizumab, paclitaxel was more homogenously distributed and mainly in actively proliferating areas of the tumor where vessels were uniformly diffused, despite paclitaxel concentration did not increase after antiangiogenic drug. No changes in paclitaxel systemic exposure was found in normal tissue. These findings suggest that a more uniform distribution of paclitaxel in the tumor of the mouse ovary is responsible for the better antitumor activity of the combination.

A.D. and I.F.N. are the recipients of a fellowship from the Italian Foundation for Cancer Research (FIRC).

Citation Format: Marta Cesca, Lavinia Morosi, Alexander Berndt, Ilaria Fuso Nerini, Alessandra Decio, Massimo Zucchetti, Raffaella Giavazzi. Bevacizumab-improved distribution of paclitaxel in ovarian cancer xenografts potentiates antitumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3377.
Abstract 346: Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies
Authors: Markovtsov Vadim V. , Lamagna Chrystelle , Chan Meagan , Yi Sothy , Young Chi , Frances Roy , Siu Stacey , Braselmann Sylvia , Li Hui , Singh Rajinder , Park Gary , Masuda Esteban , Taylor Vanessa , Payan Donald G.
Published: July 2016

Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies.

Citation Format: Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 346.
Abstract 3639: Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay
Authors: Gale Davina , Plagnol Vincent , Lawson Andrew , Pugh Michelle , Smalley Sarah , Howarth Karen , Madi Mikidache , Durham Bradley , Kumanduri Vasudev , Lo Kitty , Clark James , Green Emma , Rosenfeld Nitzan , Forshew Tim
Published: July 2016

Circulating tumor DNA (ctDNA) is becoming established as a tool to supplement conventional biopsies for molecular characterization and monitoring of solid cancers, especially for cancers where tumor tissue is difficult to obtain or is only available at limiting quantity. This requires reliable identification, in patient plasma, of tumor-specific DNA alterations that in some cases may be present as a small fraction of the total cell-free DNA molecules. To overcome these technical challenges, we have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of efficient library preparation and statistically-based analysis algorithms, this platform can be used to sequence, identify and quantify cancer mutations across a gene panel including both cancer hotspots, as well as entire coding regions of selected genes.

This poster will present validated performance specifications of this multi-gene ctDNA sequencing assay. To perform analytical validation, we used reference standards and plasma DNA controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We found that our workflow, using 4 mL input plasma, yields very high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Using dilution mixtures of well-characterised reference samples, we show that the assay accurately quantifies allele fractions with precision predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. The assay also detects DNA amplifications (including in ERBB2, MYC, KRAS, EGFR, MET, FGFR1, FGFR2) when the ctDNA are sufficiently high.

Together, these data demonstrate the analytical validity and robustness of the TAm-Seq assay and support its use as a basis for clinical applications.

Citation Format: Davina Gale, Vincent Plagnol, Andrew Lawson, Michelle Pugh, Sarah Smalley, Karen Howarth, Mikidache Madi, Bradley Durham, Vasudev Kumanduri, Kitty Lo, James Clark, Emma Green, Nitzan Rosenfeld, Tim Forshew. Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3639.
Abstract 3640: Validation of qBiomarker as an accurate and efficient mutation detection method in a comprehensive analysis of patient-derived tumor xenografts
Authors: Brait Mariana , Izumchenko Evgeny , Kagohara Luciane , Long Samuel , Wysocki Piotr , Faherty Brian , Fertig Elana , Khor Tin , Bruckheimer Elizabeth , Baia Gilson , Ciznadija Daniel , Sloma Ido , Ben-Zvi Ido , Paz Keren , Sidransky David
Published: July 2016

Screening of patients for cancer-driving mutations is now used for cancer prognosis, remission scoring and treatment selection. Although recently emerged targeted Next Generation Sequencing (NGS) - based approaches provide accurate results and offer promising diagnostic capabilities, there are still limitations which impede their adoption for large-scale population-based screening. There is a pressing clinical need for a well-validated, rapid, cost-effective and accurate mutation profiling system with optimal analytical performance in patient specimens. Given their speed, and cost-effectiveness, real-time, quantitative qPCR mutation detection techniques are well suited for the clinical environment. The novel qBiomarker somatic mutation PCR array has high sensitivity and shorter turnaround times compared to other methods. However a direct comparison to existing viable alternatives is required to assess its true potential and limitations. In this study, we extensively evaluated a panel of 117 patient-derived tumor xenografts (PDX) by the qBiomarker array and directly compared its efficacy with that of other routinely used methods for mutation detection, including Ion AmpliSeq sequencing, whole exome sequencing (WES), and ultra-sensitive droplet digital PCR (ddPCR) genotyping. Our comprehensive analysis demonstrates that qBiomarker's performance is on par with that of other routinely used but more complex, labor-intensive and expensive methods of cancer mutation detection, and provides a foundation for advancing the adoption of qBiomarker assay for cancer driving mutation profiling in clinical diagnostics. Furthermore, a large-scale direct comparison of different mutation detection approaches will lead to informed choice of screening methodologies, especially in lower budget conditions or timeframe limitations.

Citation Format: Mariana Brait, Evgeny Izumchenko, Luciane Kagohara, Samuel Long, Piotr Wysocki, Brian Faherty, Elana Fertig, Tin Khor, Elizabeth Bruckheimer, Gilson Baia, Daniel Ciznadija, Ido Sloma, Ido Ben-Zvi, Keren Paz, David Sidransky. Validation of qBiomarker as an accurate and efficient mutation detection method in a comprehensive analysis of patient-derived tumor xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3640.
Abstract 3677: Loss of IRF5 induces spontaneous murine mammary tumorigenesis
Authors: Li Dan , Bi Xiaohui , Goyeneche Corey , Pitler Amy , Barnes Betsy
Published: July 2016

Despite significant research efforts, breast cancer is still the most common malignancy in women and the most common cause of cancer death worldwide. A major challenge in breast cancer treatment is disease heterogeneity that is contributed by many factors including intrinsic cell factors, microenvironment, angiogenesis and tumor-specific immune responses. Interferon regulatory factor 5 (IRF5) is a transcription factor that controls inflammatory and immune responses. Analysis of over 3,000 human breast cancer tissues revealed that high expression of IRF5 correlates with increased survival and lower incidence of metastasis, whereas the lower quartile of IRF5 expression is a significant marker of poor prognosis for recurrence-free survival. These data support a tumor suppressor role for IRF5. To examine how loss of IRF5 expression may play a role in spontaneous mammary tumorigenesis, we generated Irf5 knockout (ko) mice in a BALB/C background. In mammary glands harvested from one-year-old female virgin ko mice (n = 15), we found an increased incidence (16%) of mammary carcinoma in situ as compared to their wild-type littermates (4%; n = 18). Of interest, the number of tumor bearing mice was significantly higher (38%) in retired ko female breeders (n = 8); however, there were no abnormalities detected in one-year-old male ko virgins or breeders (n = 6). Cellular and molecular analysis of female ko and wild-type littermates revealed that Irf5 deficiency enhanced mammary epithelial cell proliferation as indicated by an increased number of Ki67 expressing cells, and resulted in abnormal hyperbranching, which may contribute to mammary tumorigenesis. Irf5 ko breeders also showed impaired mammary gland involution after weaning, which has been shown to facilitate mammary tumor formation in humans. Taken together, these findings demonstrate that IRF5 is a gender-specific tumor suppressor in breast cancer. In this ongoing study, we are further investigating the intrinsic and extrinsic (microenvironment) role of IRF5 in mammary tumor initiation and progression.

Citation Format: Dan Li, Xiaohui Bi, Corey Goyeneche, Amy Pitler, Betsy Barnes. Loss of IRF5 induces spontaneous murine mammary tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3677.
Abstract 370: The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action
Authors: Edgar Kyle A. , Song Kyung , Schmidt Stephen , Kirkpatrick Don , Phu Lilian , Nannini Michelle , Hong Rebecca , Cheng Eric , Young Amy , Sampath Deepak , Friedman Lori S.
Published: July 2016

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha sparing inhibition of p110beta, is investigated in these preclinical studies.

Taselisib demonstrates greater potency in cancer cell lines harboring activating mutations in PIK3CA vs. wild-type lines, and induces regressions at tolerated doses in both PIK3CA mutant xenograft and patient-derived xenograft (PDX) models. When comparing taselisib to other clinical-stage PI3K inhibitors at Maximum Tolerated Dose (MTD) in vivo, taselisib confers greater activity in PIK3CA mutant models, which may indicate a larger therapeutic index. Unlike other PI3K inhibitors, taselisib has a gain of potency in PI3K alpha mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PI3K alpha mutant cells. Other clinical PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not have improved potency in PI3K alpha mutant cells due to their inability to maintain pathway suppression after alleviation of negative feedback. The unique mechanism of action of taselisib is most notable when comparing signaling suppression at 24 hours vs. 1 hour of drug exposure. In mutant cells, Taselisib displays greater pathway suppression at 24 hours and is more effective at maintaining pathway suppression upon re-activation of growth factor RTK signaling. This mechanism is specific to PIK3CA mutant cells and not observed in wildtype cells. Ongoing studies to further elucidate this mechanism of action will be presented.

Citation Format: Kyle A. Edgar, Kyung Song, Stephen Schmidt, Don Kirkpatrick, Lilian Phu, Michelle Nannini, Rebecca Hong, Eric Cheng, Amy Young, Deepak Sampath, Lori S. Friedman. The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 370.
Abstract 3996: KA2237 and KA2507: Novel, oral cancer immunotherapeutics targeting PI3K-p110β/p110δ and HDAC6 with single-agent and combination activity
Authors: Shuttleworth Stephen J.
Published: July 2016

Two de novo-designed classes of orally-active immunotherapeutics - selectively targeting the PI3K-p110β/p110δ and HDAC6 enzymes - for solid and hematological cancer treatment will be described. The former, exemplified by the clinical candidate KA2237, are uniquely-selective inhibitors of the PI3K-p110β/p110δ isoforms, displaying immunotherapeutic activity and inhibiting primary tumor growth and metastasis. The latter, for which KA2507 is the candidate compound, are HDAC6-specific inhibitors, which inhibit tumor growth through regulation of aggresome formation, and inhibition of PD-L1 expression via decrease of STAT3 phosphorylation. In both cases, selectivity has been achieved over other enzyme superfamily members, with the aim being to minimize mechanism-related toxicity, thereby potentially providing opportunities to enable elevated single dose levels - and broad combination modalities not possible with pan-inhibitors of both enzymes - to be investigated. The PI3K-p110β/p110δ and HDAC6 inhibitors display potent oral activity as single agents in in vivo tumor syngeneic models, impacting on growth inhibition, PD and tumor marker responses; the PI3K-p110β/p110δ inhibitors also inhibit metastatic spread and, following surgical removal of primary tumor, confer promising inhibition of tumor regrowth. Crucially, the inhibitors work in combination with one another, making this co-therapy approach unique in oncology, and potentially important clinically in overcoming resistance mechanisms in both solid and hematological cancer. The rationale for this combination is based upon immunotherapeutic and cell signalling mechanisms, which will be described. KA2237 is entering Phase I studies in patients with lymphoma as a single agent in Q1 2016, with KA2507 scheduled to follow in Q3 to treat multiple myeloma. Further clinical investigations into solid tumor immunotherapy - both single agent- and combination-studies - are planned.

Citation Format: Stephen J. Shuttleworth. KA2237 and KA2507: Novel, oral cancer immunotherapeutics targeting PI3K-p110β/p110δ and HDAC6 with single-agent and combination activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3996.