bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P6-05-03: Genomic diversity of ductal carcinoma in situ (DCIS) as a driver of invasion and metastasis
Authors: King LM , Marks JR , Hall AH , Temko D , Graham TA , Mardis ER , Maley CC , Hwang E
Published: May 2016

Background: Recent evidence indicates that breast cancers may have high levels of heterogeneity. Based on principles of tumor evolution, we are investigating whether DCIS with higher levels of intra-tumoral genetic heterogeneity are more likely to progress to invasive and/or metastatic disease.

Methods: Cases (DCIS with co-existing invasive or metastatic cancer) and controls (pure DCIS) are identified from Duke Pathology archives. From cases and controls, we are analyzing two areas of DCIS separated by >1cm with germ line DNA from the same subject to measure allele frequencies of somatic mutations and copy number variation (CNV). Small amounts of FFPE derived DNA are made into NGS libraries for full exome sequencing and hybridization to a 4.2 million element SNP array. Comparison of allele frequencies and CNV is made between specimens from the same cancer to assess heterogeneity.

Results: We identified a series of pure DCIS (controls for this study) and generated high coverage sequencing data from 20ng of FFPE DNA from 12 samples (4 subjects, germline + 2 DCIS containing areas from each subject) as proof of principle. We compared the occurrence of deletions, insertions and SNP's after a 20X coverage filtration. From these data, we identified between 50 to greater than 600 sequence changes in these DCIS compared to normal DNA. Present/absent calls and allele frequencies indicate both significant and variable degrees of heterogeneity even in these pure DCIS samples. Additional data is now being gathered and analyzed based on this established technical approach.

Conclusion: We have demonstrated the feasibility of acquiring high quality NGS data from archival DCIS specimens allowing us to test the hypothesis that genetic heterogeneity is a driving force in breast cancer progression. The degree and nature of this heterogeneity will be assessed in a panel of pure DCIS and DCIS co-existing with invasive and/or metastatic cancer. We are now generating and analyzing these data for Symposium presentation.

Citation Format: King LM, Marks JR, Hall AH, Temko D, Graham TA, Mardis ER, Maley CC, Hwang E. Genomic diversity of ductal carcinoma in situ (DCIS) as a driver of invasion and metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-03.
Abstract A18: Expression of the tumor suppressor gene RARRES1 in the differentiation hierarchy of breast cancer is regulated by DNA methylation
Authors: Coyle Krysta Mila , Vidovic Dejan , Dean Cheryl A. , Thomas Margaret Lois , Sultan Mohammad , Clements Derek , Vaghar-Kashani Ahmad , Wallace Melissa , Weaver Ian , Giacomantonio Carman A. , Helyer Lucy , Marcato Paola
Published: May 2016

The tumor-suppressive function of the retinoic acid (RA)-inducible gene, retinoic acid receptor responder 1 (RARRES1), has been reported in many cancer types; however, in inflammatory breast cancer, RARRES1 is pro-tumorigenic. Furthermore, high transcript levels of RARRES1 are associated with more aggressive triple-negative breast cancers (TNBCs) and poorer patient outcomes. This suggests that RARRES1 may also be oncogenic and requires investigation to determine its role in breast cancer.

Expression analyses of 20 breast cancer cell lines (including 18 TNBC and two estrogen-receptor-positive cell lines) revealed that RARRES1 is predominantly expressed in basal-like breast cancer cells, but is often methylated and silenced in claudin-low breast cancer cells. We have identified possible sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP.

Basal-like cells express higher levels of the cancer stem cell (CSC) marker ALDH1A3. Expression of RARRES1 is dependent on, and strongly correlates with ALDH1A3 expression in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum.

Finally, knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased cell proliferation and tumor growth, suggesting RARRES1 has a tumor suppressive function regardless of position on the differentiation hierarchy. We conclude that RARRES1 is a tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer.

Citation Format: Krysta Mila Coyle, Dejan Vidovic, Cheryl A. Dean, Margaret Lois Thomas, Mohammad Sultan, Derek Clements, Ahmad Vaghar-Kashani, Melissa Wallace, Ian Weaver, Carman A. Giacomantonio, Lucy Helyer, Paola Marcato. Expression of the tumor suppressor gene RARRES1 in the differentiation hierarchy of breast cancer is regulated by DNA methylation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A18.
Abstract A20: Decreased nuclear expression of the FRY protein identifies a subset of breast cancers with poor clinical outcomes
Authors: Zarbl Helmut , Graham Jessica C. , Takizawa Norio , Fang Mingzhu , Gong Zhihong , Estrella Brian , Ren Xuefeng
Published: May 2016

The goal of the present study was to determine if decreased nuclear expression of the human FRY protein can serve as a biomaker of breast cancer progression and outcomes. We previously identified the rat Fry gene as a putative mammary carcinoma susceptibility (Mcs) gene. We further demonstrated that FRY expression was decreased in several human breast cancer cell lines. Moreover, ectopic expression of wildtype Fry suppressed tumorigenicity of the triple negative MDA-MD-231 breast cancer cells in vitro and in vivo by promoting epithelial cell differentiation. To evaluate the contribution of altered FRY expression to the clinical progression of human breast cancer, we first compared FRY mRNA expression in >4,800 clinically annotated human breast cancers using DNA microarray data from 19 distinct cohorts available from the Oncomine 3.0 Cancer Profiling Database. The analysis indicated that decreased FRY mRNA was significantly correlated with poorly differentiated tumor histopathology, increased Elston tumor grade, and triple negative status (loss of estrogen, progesterone and Her2 receptors). Using commercially available breast cancer tissue microarrays, we used semi-quantitative immunohistochemistry and quantitative image analysis to compare FRY protein expression in normal mammary tissue and tumors. The results indicated that loss of FRY expression was a frequent event during progression of mammary carcinomas. We therefore analyzed >1200 clinically annotated breast cancers represented on tissue microarrays from commercial suppliers and the National Cancer Institute (NCI) Cancer Diagnosis Program, including the Breast Cancer Diagnosis and Progression panels. FRY protein expression was evaluated by immunohistochemical staining and was graded on a scale of 0 to 4 (0, +, ++, +++, ++++). The analysis indicated that decreased nuclear expression of the FRY protein was significantly correlated with poorly differentiated histopathologies, increasing Elston grade and loss of the estrogen receptor. Tumors with decreased FRY expression were also more likely to metastasize and were associated with decreased tumor-free survival. These findings indicated that loss of nuclear FRY expression identifies a subset of breast cancer with poor clinical outcomes, suggesting that FRY may serve as an early biomarker for breast cancer progression and prognosis.

Citation Format: Helmut Zarbl, Jessica C. Graham, Norio Takizawa, Mingzhu Fang, Zhihong Gong, Brian Estrella, Xuefeng Ren. Decreased nuclear expression of the FRY protein identifies a subset of breast cancers with poor clinical outcomes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A20.
Abstract A40: ESR1 gene alterations in the metastatic breast cancer with resistance to aromatase inhibitor
Authors: Iwase Hirotaka , Takeshita Takashi , Yamamoto-Ibusuki Mutsuko , Inao Touko , sueta Aiko , Fujiwara Saori
Published: May 2016

Background: Estrogen receptor (ER) positive breast cancer can be treated by endocrine therapy; however a certain population of ER-positive patients becomes resistant to endocrine therapy after long-term estrogen deprivation treatment. ESR1 gene alterations, mutations, translocation and amplification, have been reported as one of the reasons.

Patients and Methods: We successfully obtained metastatic tissue samples from 30 patients with resistance to aromatase inhibitor (AI), and 7 patients with resistance to ethinylestradiol (EE2). ER and PgR expression by immunohistochemistry, and ESR1 gene amplification using FISH and copy number variation (CNV) by PCR, and ESR1 gene mutation including Y537N, Y537C, Y 537S and D538G by droplet digital PCR, were examined, in order to evaluate the endocrine responsiveness.

Results: High expression of ER (median; 90%) and low expression of PgR (median 5%) were seen in majority of the patients treated by AI. Signal enhancement of DNA-FISH (> 2.0 fold) was seen in 2 (8%) of 25 patients, and signal gain (1.0< ≤2.0 fold) was seen in 11 (44%) of 25 patients. The increasing of DNA-CNV of ESR1 gene was seen in 3 cases of the 25 examined cases. There was no relationship between DNA-FISH and CNV of ESR1. High frequent ESR1 mutations were seen in 7 (28%) at Y537N, 4 (16%) at Y537C, 11 (44%) at Y 537S, and 2 (8%) at D538G. Double mutation, Y537N/S, was seen in 6 (24%) patients, who tended to unresponsive to subsequent endocrine therapy. After EE2 treatment, ER expression was decreased and PgR expression was increased, but there was no relationship between EE2 treatment and ESR1 gene alteration.

Conclusion: In the patients with resistance to AI, high ER / low PgR expression was frequently seen, which was no correlation to ESR1 DNA-amplification. The long non-coding RNA in ESR1 gene locus might be related to this discrepancy (Tomita et al; Nat Commun. 2015). Furthermore, accumulation of ESR1 gene mutations tended to be related to endocrine unresponsiveness.

Citation Format: Hirotaka Iwase, Takashi Takeshita, Mutsuko Yamamoto-Ibusuki, Touko Inao, Aiko sueta, Saori Fujiwara. ESR1 gene alterations in the metastatic breast cancer with resistance to aromatase inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A40.
Abstract A59: Prognostic relevance of PTEN and TP53 deletion in breast cancer
Authors: Kluth Martina , Lebeau Anette , Witzel Isabell , Mahner Sven , Woelber Linn , Geist Stefan , Paluchowski Peter , Wilke Christian , Heilenkoetter Uwe , Mueller Volkmar , Simon Ronald , Sauter Guido , Terracciano Luigi , Krech Rainer , Assen Albert von der , Burandt Eike , Lebok Patrick
Published: May 2016

Inactivation of PTEN and TP53 have been suggested to promote unfavorable phenotypes in various cancers, including breast cancer. Chromosomal deletion is one well established mechanism for inactivation of these genes. In breast cancer, deletions of TP53 and PTEN are known to occur, but systematic analyses of its clinical relevance are lacking. We thus analyzed a tissue microarray (TMA) with 2,197 breast cancers by fluorescence in-situ hybridization (FISH) using specific probes against TP53 and PTEN in combination with respective centromere probes. We found, that TP53 deletion (29%/1026 cancers) occur more frequent in breast cancers than deletions of PTEN (19%/1,239 cancers). The frequency of PTEN deletion varied between histological subtypes with highest deletion rate in medullary cancers (18 of 39, 46%) and lowest deletion rate in mucinous cancers (1 of 28; 3%; p<0.0001) for mucinous vs. medullary). PTEN deletions were significantly linked to various unfavorable tumor features, including advanced tumor stage (p=0.0054), high-grade (p<0.0001), high tumor cell proliferation (Ki67 Labeling Index; p<0.0001), and shortened overall survival (p=0.0090). Deletions of TP53 were significantly linked to high-grade (p<0.0001) and high tumor cell proliferation (p=0,0193) but unrelated to histological subtypes, tumor stage, nodal status, and patient survival. Deletion of PTEN and TP53 were unrelated to each other (p=0.6469). Combined analysis of PTEN and TP53 deletions showed that TP53 deletion had no additional effect on patient prognosis in cancers with or without PTEN deletion. In conclusion, our data support a role of PTEN and TP53 deletions in breast cancer. However, only PTEN deletion is inked to aggressive tumor behavior, while TP53 deletion has no prognostic relevance in breast cancer.

Citation Format: Martina Kluth, Anette Lebeau, Isabell Witzel, Sven Mahner, Linn Woelber, Stefan Geist, Peter Paluchowski, Christian Wilke, Uwe Heilenkoetter, Volkmar Mueller, Ronald Simon, Guido Sauter, Luigi Terracciano, Rainer Krech, Albert von der Assen, Eike Burandt, Patrick Lebok. Prognostic relevance of PTEN and TP53 deletion in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A59.
Abstract A61: Cell cycle regulated centrosome orientation during directed migration requires Aurora Kinase A
Authors: Chu Tony LH , Won Jennifer , Nemirovsky Oksana , Fotovati Abbas , Nielsen Torsten , Maxwell Christopher A
Published: May 2016

Cell cycle progression and the acquisition of a migratory phenotype are hallmarks of human carcinoma cells. Here we report a strong correlation between cell cycle progression into G2 phase and an increased migratory velocity for non-malignant, immortalized mammary epithelia nMuMG (Mus musculus mammary gland) cells, or malignant cervical carcinoma HeLa cells, expressing a fluorescent ubiquitin cell cycle indicator and quantified in wound closure assays with high content multi-parameter live cell imaging. Cells at the wound edge exhibited elevated microtubule nucleation capacity at centrosomes, and a reduction of this capacity through inhibition of aurora kinase A was sufficient to impair migration velocity without affecting cell cycle dynamics. In migratory cells, active aurora kinase A at the centrosome correlated with nucleation capacity. In clinically annotated mammary carcinoma tissues (n=3,992), aurora kinase A activity, as assessed by the abundance of phosphorylated-RHAMM (Receptor for Hyaluronan Mediated Motility) was a significant predictor of breast cancer specific survival and relapse free survival in patients with estrogen receptor negative breast cancer (n= 941), triple negative phenotype breast cancer (n= 538) or basal-like subtype breast cancer (n= 293), but not in those patients with estrogen receptor positive breast cancer (n= 2,218). In estrogen receptor negative tumors from patients that received no adjuvant systemic therapy (n= 453), the levels of phosphorylated-RHAMM associated with the presence of distal nodal metastasis and predicted a subset of patients at high risk of recurrence. Together, these data identify aurora kinase A activity as a regulatory pathway that intersects cell cycle progression and cell migration, with potential to influence the metastasis of estrogen receptor negative breast tumors and patient survival.

Citation Format: Tony LH Chu, Jennifer Won, Oksana Nemirovsky, Abbas Fotovati, Torsten Nielsen, Christopher A Maxwell. Cell cycle regulated centrosome orientation during directed migration requires Aurora Kinase A. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A61.
Abstract A72: The E3-ligase E6AP represses breast cancer metastasis through regulation of ECT2-Rho-GTPases signaling
Authors: Mansour Mariam , Haupt Sue , Chan Ai-Leen , Godde Nathan , Rizzitelli Alexandra , Loi Sherene , Caramia Franco , Deb Siddartha , Takano Elena A. , Bishton Mark , Johnstone Cameron , Levav-Cohen Yarra , Jiang Yong-Hui , Yap Alpha S. , Fox Stephen , Bernard Ora , Anderson Robin , Haupt Ygal
Published: May 2016

Metastatic disease is the major cause of breast cancer related death and despite many advances, current therapies are rarely curative. Tumor cell migration and invasion require actin cytoskeletal reorganisation, to endow cells with capacity to disseminate and initiate the formation of secondary tumors. However, it is still unclear how these migratory cells colonise distant tissues to form macrometastases. The E6-associated protein, E6AP, acts both as an E3 ubiquitin-protein ligase and as a coactivator of steroid hormone receptors. We report that E6AP suppresses breast cancer invasiveness, colonisation and metastasis in mice and in breast cancer patients, loss of E6AP associates with poor prognosis, particularly for with basal breast cancer. E6AP regulates actin cytoskeletal remodelling via regulation of Rho-GTPases, acting as a negative regulator of ECT2, a GEF required for activation of Rho-GTPases. E6AP promotes ubiquitination and proteasomal degradation of ECT2 for which high expression predicts poor prognosis in breast cancer patients. We conclude that E6AP suppresses breast cancer metastasis by regulating actin cytoskeleton remodelling through the control of ECT2 and Rho-GTPase activity. These findings establish E6AP as a novel suppressor of metastasis and provide a compelling rationale for inhibition of ECT2 as a therapeutic approach for patients with metastatic breast cancer.

Note: This abstract was not presented at the conference.

Citation Format: Mariam Mansour, Sue Haupt, Ai-Leen Chan, Nathan Godde, Alexandra Rizzitelli, Sherene Loi, Franco Caramia, Siddartha Deb, Elena A. Takano, Mark Bishton, Cameron Johnstone, Yarra Levav-Cohen, Yong-Hui Jiang, Alpha S. Yap, Stephen Fox, Ora Bernard, Robin Anderson, Ygal Haupt. The E3-ligase E6AP represses breast cancer metastasis through regulation of ECT2-Rho-GTPases signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A72.
Abstract B11: A dual PI3K/mTOR inhibitor, BEZ235 blocks tumor-induced angiogenesis: Evidence for an effect on the tumor and endothelial compartment
Authors: De Pradip , Sun Yuliang , Carlson Jennifer H. , Lin Xiaoqian , Dey Nandini , Leyland-Jones Brian
Published: May 2016

Background: Tumor progression including HER2+ breast tumor, particularly in aggressive and malignant tumors, is associated with the angiogenesis. To investigate the effects of BEZ235 on angiogenesis, we evaluated its effects on the hypoxic stabilization or the PI3K-AKT-mTOR pathway-mediated synthesis of hypoxia-inducible factor -1α (HIF-1α) and tumor-induced angiogenesis. Methodology: Here we have studied the in vitro and in vivo effects of BEZ235 in HER2+/T-sensitive (BT474), HER2+/T-resistant (BT474HerR) and HER2+/PIK3CA (HCC1954 & UACC893) mutated models. We assessed in vitro activation status of the PI3K-AKT-mTOR signaling pathway following BEZ235 treatment in HER2+ BC cell lines. We next evaluated the impact of BEZ235 on hypoxia-induced or the PI3K-AKT-mTOR pathway ⊠mediated HIF1α stabilization/synthesis which is a master regulator of angiogenesis and also tumor-induced angiogenesis using xenograft models. Results: The results show that 1) BEZ235 inhibited downstream activation of the PI3K-AKT-mTOR signaling pathway effectors, p-AKT (Ser473, The308), p-P70S6K, p-S6RP and p-4EBP1, 2) unlike other pan-PI3Kinase inhibitor (e.g. LY294002, SF1126), BEZ235 has no effect on hypoxia-mediated stabilization of HIF1α however, heregulin-induced HIF1α synthesis which is an important angiogenic modulator in breast cancer cells, was significantly decreased following the treatment of BEZ235 in HER2+ and HER2+/PIK3CA mutated breast cancer cells, 3) interestingly, BEZ235 mediated abrogation of HIF1α synthesis is more pronounced than mTORC1 inhibition alone, 4) integrin-directed endothelial cell migration is one of the critical steps for tumor-induced angiogenesis. BEZ235 abrogates endothelial cells functions especially integrin-directed HUVEC cells migration on vitronectin (avb3/ avb5) and significantly abrogates endothelial cells tube formation on Matrigel and 5) furthermore, we evaluated the effects of BEZ235 on the capacity of HER2+ and HER2+/PIK3CA mutated breast tumor cells to recruit a blood supply in vivo during thrice weekly treatments with BEZ235. A microvessel density (MVD) in control versus BEZ235-treated tumors showed a significant decrease in MVD (CD31+) and also VEGFR immunostaining in BEZ235 treated tumors. Conclusion: Our data suggest that BEZ235 has potent antiangiogenic activity in three different xenograft models probably via mTOR-HIF1α-VEGF signaling axis.

Citation Format: Pradip De, Yuliang Sun, Jennifer H. Carlson, Xiaoqian Lin, Nandini Dey, Brian Leyland-Jones. A dual PI3K/mTOR inhibitor, BEZ235 blocks tumor-induced angiogenesis: Evidence for an effect on the tumor and endothelial compartment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B11.
Abstract B26: Identification of genes that predict response to paclitaxel in breast cancer using an in vivo genome-wide knockdown screen
Authors: Sultan Mohammad , Huynh Thomas Tan , Thomas Margaret Lois , Coyle Krysta Mila , Giacomantonio Carman A. , Marcato Paola
Published: May 2016

Treatment decisions for breast cancer are based upon stage, tumor grade and hormone receptor status, and can include surgical resection, hormone receptor antagonists, radiation, and chemotherapy (e.g. paclitaxel). Breast cancer treatment success depends upon avoidance of chemotherapy resistance (i.e. achieving complete response) and prevention of both over- and under-treatment. Increased understanding of the genes which cause resistance and sensitivity to currently used drugs would lead to development of more effective therapeutic strategies that are specifically tailored to patient groups based on molecular profiling of their tumors (i.e. personalized medicine). Being able to identify the genes which when expressed in a tumor predict sensitivity or resistance to treatment prior to administration of paclitaxel would improve treatment efficacy and patient survival. We performed an in vivo shRNA genome-wide screen with MDA-MB-231 tumors treated with paclitaxel for the purpose of identifying genes which determine breast cancer response to paclitaxel. Completion of 6 replicates of the in vivo screen identified 26 putative paclitaxel sensitivity genes and 14 putative paclitaxel resistance genes (e.g. BCL6) for breast cancer. Screen-identified putative paclitaxel resistance were verified by individual knockdown clone generation and comparison of their sensitivity to paclitaxel-induced decreased cell proliferation, cell-cycle arrest, and apoptosis to a shRNA scramble control clone. Upon individual knockdown of the putative resistance genes (e.g. BCL6), MDA-MB-231 cells were more sensitive to paclitaxel and demonstrated increased apoptosis and decreased paclitaxel IC50 concentrations. Finally, expression of a preliminary gene signature generated from the screen-identified hits was tested for its ability to predict response to paclitaxel in two archived patient data sets. The preliminary gene signature predicted response to paclitaxel in the datasets with an accuracy ranging from 70 to 100%. Further confirmation experiments of the remaining potential resistance and sensitivity genes will help to generate a more robust genetic profile which can be used to identify candidate breast cancer patients who would most benefit from paclitaxel treatment as opposed to treatment with other drugs.

Citation Format: Mohammad Sultan, Thomas Tan Huynh, Margaret Lois Thomas, Krysta Mila Coyle, Carman A. Giacomantonio, Paola Marcato. Identification of genes that predict response to paclitaxel in breast cancer using an in vivo genome-wide knockdown screen. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B26.
Abstract B43: APOBEC3B upregulation by the PKC-NFκB pathway in breast cancer
Authors: Leonard Brandon , McCann Jennifer , Starrett Gabriel , Kosyakovsky Leah , Amy Molan , Burns Michael , McDougle Rebecca , Parker Peter , Brown William , Harris Reuben
Published: May 2016

Overexpression of the antiviral DNA cytosine deaminase APOBEC3B has been linked to somatic mutagenesis in breast and other cancer. Human papillomavirus (HPV) infection accounts for APOBEC3B upregulation in cervical and head/neck cancers. However, the responsible mechanisms are unclear for non-viral malignancies such as breast cancer. Here, we demonstrate APOBEC3B upregulation through the PKC-NFκB pathway. PKC activation by the diacylglycerol mimic PMA causes specific and dose-responsive increases in APOBEC3B mRNA, protein, and activity levels, which are strongly suppressed by PKC and NFκB inhibition. Induction correlates with RELB (but not RELA) recruitment to the endogenous APOBEC3B gene implicating non-canonical NFκB signaling. Relevance to tumors is supported by PKC inhibitor-mediated APOBEC3B downregulation in multiple breast cancer cell lines. These data establish the first mechanistic link between APOBEC3B and a common signal transduction pathway, suggesting that existing PKC-NFκB inhibitors could be repurposed to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes such as drug resistance and metastases.

Citation Format: Brandon Leonard, Jennifer McCann, Gabriel Starrett, Leah Kosyakovsky, Molan Amy, Michael Burns, Rebecca McDougle, Peter Parker, William Brown, Reuben Harris. APOBEC3B upregulation by the PKC-NFκB pathway in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B43.
Abstract B68: Contributions of STAT3 to pro-tumorigenic alterations within the microenvironment during mammary tumorigenesis
Authors: Chuntova Polly , Bohrer Laura R. , Schwertfeger Kaylee L.
Published: May 2016

Inflammation has long been recognized as a possible trigger that contributes to tumor initiation. Numerous studies have shown that cells of both the innate and adaptive immune systems are able to support tumor formation and growth through secretion of growth factors and cytokines and by actively shielding the tumor from wider immune surveillance. As a result, inflammation is generally linked to worse patient outcome. However, depending on factors such as tumor size, extracellular matrix, and the complex composition of immune cell infiltrates, inflammation can promote both pro- and anti-tumor properties. Our studies focus on identifying novel mechanisms through which tumor-derived cytokines act in a paracrine manner to regulate the functions of infiltrating inflammatory cells in the tumor microenvironment. We have previously shown that activation of the fibroblast growth factor receptor-1 (FGFR1) oncogene results in the production of high levels of interleukin 6 (IL-6) family members by mammary epithelial cells. Histological examination of FGFR1-driven tumors revealed high levels of STAT3 activation in both tumor cells and macrophages. Thus, further studies focused on identifying mechanisms through which STAT3 activation in different cellular compartments contributes to mammary tumor growth. Initial studies focused on the consequences of STAT3 activation in epithelial/tumor cells. We found that epithelial cell STAT3 activation resulted in accumulation of the extracellular matrix component hyaluronan, which contributed to epithelial cell proliferation, survival and migration. In macrophages, STAT3 is the transcription factor required for transduction of the anti-inflammatory response initiated by interleukin-10. Ongoing experiments have revealed that in primary STAT3fl/fl cre+ bone marrow-derived macrophages, absence of STAT3 signaling reveals a profoundly enhanced pro-inflammatory phenotype. In vitro, treatment of macrophages lacking STAT3 with recombinant IL-6 results in prolonged activation of the proinflammatory STAT1, as well as upregulation of inflammatory molecules such as TNFα and CCL2. Ongoing experiments are aimed at evaluating the functional consequences of this altered tumor microenvironment in vivo during tumor initiation and growth. As anti-STAT3 therapies have began entering clinical trials for solid tumors (including breast tumors), understanding the mechanisms through which STAT3 promotes tumor growth and progression is critical for developing optimal therapeutic strategies.

Citation Format: Polly Chuntova, Laura R. Bohrer, Kaylee L. Schwertfeger. Contributions of STAT3 to pro-tumorigenic alterations within the microenvironment during mammary tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B68.
Abstract IA01: Potential applications of genomics in patients with metastatic breast cancer
Authors: André Fabrice
Published: May 2016

Genomic studies have shown that breast cancer includes large number of genomic segments. From this observation has arisen the concept that sequencing could be used to select breast cancer patients for new therapies. There are at least six applications of genomic tests to individualize therapy in breast cancer. In the present document and presentation, we will only focus on metastatic breast cancers.

First, genomic tests can be used to identify oncogenic drivers at the individual level. There are seven genomic alterations that are currently the focus on genomic-driven drug development. This includes AKT1 mutations, ERBB2 mutations, PIK3CA mutations, FGFR1 amplification, CCND1 amplification, BRCA1/2 mutations and PTEN mutations or loss. With the exception of CCND1, early phase trials have suggested that these genomic alterations could be associated with antitumor activity when targeted with the appropriate compound. Beyond the driver identification at the DNA level, some are developing RNA-based assays to identify pathway activation in individuals. As illustration, gene expression could assess mTOR, CDK4, AR, ER activations. Whether the assessment of pathway activation identifies patients eligible for targeted therapies is not yet proven.

The second application of genomics is the identification of genomic alterations involved in drug resistance. As illustration, it has been shown that ESR1 and PTEN mutations could be involved in the resistance to endocrine therapy or PI3K inhibitors. ctDNA could be the perfect tool to monitor appearance of these genomic alterations and treat patients accordingly.

The third application of genomics could be the identification of patients who are highly sensitive to immunotherapeutics. Several papers have shown that high mutation load and neoantigens could be associated with sensitivity to immune checkpoint inhibitors. During the conference, we will share some preliminary data on the mutational load and neoantigens in metastatic samples of breast cancer patients.

Genomics could also be used to identify mutational processes and DNA repair defect.

Finally, genomic assays could allow identification of patients with metastatic breast cancer who present a very poor outcome. Such assay could be used to identify a population of patients where fast track drug approval is needed.

Clinical trials are ongoing to determine whether the use of multigene assay could improve outcome in metastatic breast cancer patients, and which genes are associated with drug efficacy.

Citation Format: Fabrice André. Potential applications of genomics in patients with metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA01.
Abstract IA06: Spatial systems biology of cancer
Authors: Gray Joe
Published: May 2016

International efforts have now defined the genomic landscapes of most major human cancer types. The genomic landscape of breast cancer is particularly well described in several recent publications. The high level view of this disease suggests a remarkable level of inter- and intra-tumor heterogeneity, the presence of a few recurrent and many rare genomic aberrations and substantial genomic and epigenomic heterogeneity. The challenge now is to understand how these aberrations and tumor extrinsic influences from the microenvironment collaborate to deregulate aspects of cell differentiation, proliferation, apoptosis, DNA repair, senescence, motility and multiscale architecture that include cancer progression and response to therapy. To this end, the OHSU Center for Spatial Systems Biomedicine has deployed a suite of measurement technologies that enable high throughput assessment of changes in these endpoints to siRNA knockdown of genes found by the TCGA and international genomics efforts to be aberrant and/or involved in regulatory networks that are associated with differentiation subtype and/or differential response to therapy. These studies have revealed both recurrent and rare aberrations that strongly influence these endpoints as well as a range of molecular architectures that influence aspects of receptor tyrosine kinase signal transduction. This presentation will summarize the results of the siRNA knockdown experiments and focus on the concept that analysis of the architectural features influenced by genomic and epigenomic abnormalities will suggest new therapeutic approaches. Illustrative examples of architectural features revealed using advanced imaging approaches that suggest new approaches to the design of RTK targeted therapies include (a) Ras/Raf multimers associated with signal transduction, (b) actin associated microfilaments involved in ERBB2 signal transduction, and (c) intratumor heterogeneity in differentiation status that influences responses to a range of kinase inhibitors.

Citation Format: Joe Gray. Spatial systems biology of cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA06.
Abstract IA18: Development of targeted therapeutics using patient-derived xenografts
Authors: Lewis Michael T.
Published: May 2016

Clinically, breast cancers are divided into three distinct groups: those that express the estrogen hormone receptor (ER+) (which typically also express the progesterone hormone receptor (PR+), those that are amplified or overexpress the ErbB2 (HER2) oncogene (HER2+), and those that express none of these three markers (termed triple negative breast cancer TNBC). Unlike ER+ and HER2+ breast cancers, there are currently no targeted therapies against TNBC. Thus, the development of targeted therapies for TNBC is an active area of research.

Entry into the clinical trial pipeline is a major milestone in the process of drug development. However, human clinical trials have several inherent limitations including high cost and long time horizon. Add to these limitations the fact that many promising targeted therapeutics enter into this pipeline without detailed knowledge of the patient population most likely to respond, and without knowledge of particular drug combinations that might be more efficacious than others. As a consequence, the inherent limitations combined with lack of knowledge leads to a high frequency of failure, with failure late in the process costing extraordinary amounts of time and money.

In theory, many of the limitations of human trials can be overcome using an animal clinical trial platform that exploits collections of patient-derived xenograft (PDX) models that recapitulate much of the biology of the tumor of origin in the patient.

Our laboratory has developed a large collection of human breast cancer PDX with which we are evaluating a number of candidate targeted therapeutics alone and in various combinations, with a particular focus on overcoming chemotherapy resistance in TNBC. We show that animal clinical trials are feasible, and can be conducted efficiently, using minimal staff. Further, we demonstrate that results in animal clinical trials can be used to inform clinical trial design. We anticipate that insertion of animal clinical trials early in the drug development pipeline can streamline the drug development process and lead to more efficient evaluation of drug efficacy, saving both time and money. Ultimately, if robust genetic, epigenetic, or molecular predictors of differential treatment response can be identified in PDX models and applied clinically, it may be possible to avoid ineffective treatments, minimize exposure to cytotoxic agents, and ultimately increase survivorship.

Citation Format: Michael T. Lewis. Development of targeted therapeutics using patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA18.
Abstract IA25: Deep sequencing of circulating tumor DNA for personalized cancer detection and monitoring
Authors: Diehn Max
Published: May 2016

Circulating tumor DNA (ctDNA) represents a promising biomarker for sensitive, specific, and dynamic detection of disease burden in cancer patients. Additionally, ctDNA analysis allows non-invasive access to cancer genomes and therefore can be used for non-invasive tumor genotyping and monitoring of resistance mutations. In this presentation I will describe technical aspects of ctDNA detection and emerging clinical results from recent studies employing ctDNA analysis.

Citation Format: Max Diehn. Deep sequencing of circulating tumor DNA for personalized cancer detection and monitoring. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA25.
Abstract A22: Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis
Authors: Carr-Wilkinson Jane , Prathalingam Nilendran , Pal Deepali , Forgham Helen , Lako Majlinda , Herbert Mary , Tweddle Deborah A.
Published: May 2016

Background and Aims: Neuroblastoma is an embryonal malignancy derived from neural crest cells which give rise to the sympathetic nervous system (SNS). One of the main therapeutic challenges in neuroblastoma continues to be the emergence of relapsed disease, which occurs in 50% of patients with high risk disease and is associated with resistance to therapy. Our aim was to differentiate human embryonic stem cells (hESCs) to sympathetic neurons (SN) to model normal human SNS development.

Results: Using the stromal-derived inducing activity (SDIA) of murine PA6 cells in combination with BMP4 and B27 neuronal supplement, H9 and a newly derived hESC line Ncl(R)14, were induced to differentiate to neural crest stem cells and SN. After 7 days of PA6 cell co-culture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker Peripherin increased. Q-RT-PCR showed that expression of the pluripotency marker OCT 4 decreased, whereas p53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A marked increase in expression of the catecholaminergic marker Tyrosine Hydroxylase (TH) and the noradrenergic marker Dopamine Beta Hydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH and Peripherin. SN were identified by immunofluorescence by co-expression of TH & Peripherin or DBH & PHOX2B in p75+ cells. Live cell analysis and imaging showed increased migration in p75+ cells compared with p75- cells, consistent with a migratory neural crest phenotype in-vitro.

Conclusions: We have established a model of nor-adrenergic SNS development using two hESC lines to improve our understanding of normal human SNS development and in future studies the pathogenesis of neuroblastoma.

Citation Format: Jane Carr-Wilkinson, Nilendran Prathalingam, Deepali Pal, Helen Forgham, Majlinda Lako, Mary Herbert, Deborah A. Tweddle. Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A22.
Abstract A25: Role of ABCB5 in progression and therapeutic resistance of glioblastoma
Authors: Banerjee Pallavi , Berg Gretchen , Wilson Brian J. , Guo Qin , Frank Markus H. , Frank Natasha Y.
Published: May 2016

Glioblastoma multiforme (GBM), one of the most lethal human cancers, is the most common and aggressive primary malignant brain tumor. Poor prognosis and high lethality result from tumor recurrence and extreme resistance to chemotherapeutic treatment. Compelling evidence suggests that GBM is a heterogeneous tumor composed of bulk tumor cells as well as of subpopulations of slow-proliferating cancer stem cells with tumor-initiating potential, which evade cell killing and are responsible for tumor recurrence. Current therapeutic strategies are thus aimed at eliminating these refractory tumor subpopulations to alleviate chemoresistance and recurrence of GBM. ATP-binding cassette member B5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies, including malignant melanoma, hepatocellular carcinoma and colorectal cancer. Moreover, ABCB5 has been shown to functionally contribute to tumor progression and therapeutic resistance of human cancers. In the current study, we examined the potential role of ABCB5 in malignant growth and chemoresistance of GBM. We found ABCB5 to be co-expressed with GBM stem cell marker CD133 in all of three human GBM cell lines tested thus far, i.e. U87 MG, LN18 and LN229. Functional blockade of ABCB5 using an anti-ABCB5 monoclonal antibody (mAb) (clone 3C2-1D12) inhibited proliferation and survival of human GBM cells, and sensitized GBM cultures to Temozolomide (TMZ)-induced cell killing and apoptosis. Likewise, in vivo systemic administration of anti-ABCB5 mAb to immunodeficient (NSG) mice bearing human GBM xenografts resulted in significant reduction of tumor growth and sensitization of GBM tumors to TMZ therapy, underscoring the potential of ABCB5-targeting strategies for reversal of GBM chemotherapeutic resistance. Mechanistically, we found that ABCB5 blockade significantly altered molecules involved in cell cycle regulatory checkpoints to revoke TMZ-induced G2/M arrest, explaining how treatment with ABCB5 mAb is augmenting drug-mediated apoptosis. Overall, our data reveal a novel role of ABCB5 as a marker of GBM chemoresistance and show that functional ABCB5 blockade inhibits GBM tumor growth in vivo. These findings underline the potential clinical relevance and therapeutic importance of ABCB5 targeting as a novel therapeutic approach to GBM eradication.

Citation Format: Pallavi Banerjee, Gretchen Berg, Brian J. Wilson, Qin Guo, Markus H. Frank, Natasha Y. Frank. Role of ABCB5 in progression and therapeutic resistance of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A25.
Abstract A29: JNK contributes to BMP loss induced intestinal tumorigenesis in Drosophila
Authors: Zhou Jun , Boutros Michael
Published: May 2016

Genetic instability and mutations are contributed to colon cancer development. For instance, loss of intestinal BMP signaling through mutation on SMAD4 and BMPR1A is a prominent cause of gastrointestinal cancers. The BMP pathway functions as a negative regulator of crypts and is taken to be essential for terminal differentiation. However the genetic causes and molecular mechanism of BMP loss induced tumorigenesis remains largely unknown. Here, we used Drosophila intestinal tumors generated by suppressing BMP/Dpp signaling as a model to investigate the genetic mutations are essential for tumor development. We found BMP loss induced Drosophila intestinal tumor displays cellular heterogeneity is similar to mammalian colon tumor, whereas Drosophila intestinal tumor contains stem cell, its progenitor cells (enteroblast), immatured enterocytes and enteroendocrine cells. In addition, we observed signaling molecules significantly upregulated in BMP defective tissue such as, STAT ligands (Upd, Upd2, 3), EGFR ligands (Spitz, Vein), Insulin receptor and JNK components (dFos, Puc). Through genetic gain and loss of function experiments, we showed JNK activity contributes to the BMP loss induced intestinal tumorigenesis. We observed high JNK activity inside BMP defective tumor using clonal analysis. The growth of BMP defective tumor can be strongly suppressed by JNK inhibition through genetic suppression (dFOS RNAi) or compounds feeding (SP600125 inhibitor). Furthermore, JNK activation through herozygous mutation in its negative regulator Puckered enhanced the BMP loss induced tumorigenesis and lethality. Hence, our data identify JNK activity is essential for the development of BMP defective intestinal tumor.

Citation Format: Jun Zhou, Michael Boutros. JNK contributes to BMP loss induced intestinal tumorigenesis in Drosophila. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A29.