bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-381: Effects of sequencing parameters and panel size on mutational burden calculations
Authors: Yourstone Scott , Mola Natalie , Weigman Victor J.
Published: August 2018

Immunotherapies are an emerging approach for cancer treatment that leverage the power of the immune system to attack cancer cells. By targeting immune check-point pathways such as PD-1, these treatments have been highly effective for some patients diagnosed with skin, bladder, lung, or kidney cancer. However, response rates for these treatments vary, making it beneficial to identify patients who are likely to respond to the treatment. Qualitative methods for predicting immunotherapy response are available but are technically challenging and subjective. An emerging quantitative predictor of immunotherapy response, tumor mutational burden (TMB), measures the number of somatic mutations per Mb of the genome. TMB calculations using whole exome data have been shown to correlate with patient response to immunotherapies. However, derivation of this score is not well characterized, nor is the score universally performed across laboratories. Here we describe the results from calculating TMB scores varying cancer panel size and amount of sequencing generated. First, variants are called in matched tumor and normal samples within Exomes. Then low coverage variants are removed along with variants outside the targeted, high-confidence regions in each panel. The remaining variants in the normal samples constitute the germline variants and are subtracted from the number of variants identified in tumor samples to yield the TMB score. We then repeated this across multiple indications across TCGA to review robustness of score in indications across the spectra of burden. TMB scores from the our comprehensive cancer closely resembles scores calculated using whole exome data across indications. We propose rules for gene content and sequencing depth for providing a range of burden scores that could be used for determining thresholds needed for determining therapeutic implications. We suggest that only a small subset of genes are required for accurate TMB estimation, thus reducing costs associated with predicting patient response to immunotherapy.

Citation Format: Scott Yourstone, Natalie Mola, Victor J. Weigman. Effects of sequencing parameters and panel size on mutational burden calculations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-381.
Abstract LB-395: STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells contributes to the malignant progression of gastric cancer cells
Authors: Chen Jinfei , Yang Fen
Published: August 2018

BACKGROUND: Long noncoding RNAs (lncRNAs) are an important class of functional regulators involved in gastric cancer (GC). Studying aberrantly expressed lncRNAs may provide us with new insights into the occurrence and development of gastric cancer by acting as oncogenes or tumor suppressors. In this study, we aim to examine the expression pattern of lncRNA HAGLROS in GC and its biological role and mechanisms in tumor progression.

METHODS: Bioinformatics analysis and qRT-PCR were performed to detect the relative expression of HAGLROS in GC tissues and cell lines. Gain or loss of function approaches were used to investigate the biological functions of HAGLROS. The effect of HAGLROS on proliferation was evaluated by MTT, colony formation assay and nude mouse xenograft model. Wound healing and Transwell assays were used to study the invasion and migration of GC cells. FISH, RIP, RNA-seq, Luciferase report assays, RNA pulldown and Western blot were fulfilled to measure molecular mechanisms. Results are shown as means ± S.D. and differences were tested for significance using Student's t-test (two-tailed).

RESULTS: We screened out HAGLROS, whose expression was significantly increased and correlated with outcomes of GC patients by publicly available lncRNAs expression profiling and integrating analyses. Exogenous down-regulation of HAGLROS expression significantly suppressed the cell proliferation, invasion and migration. Mechanistic investigations showed that HAGLROS was a direct target of transcriptional factor STAT3. Moreover, HAGLROS knockdown decreased mTOR expression and increased autophagy-related genes ATG9A and ATG9B expression. Further investigation showed that HAGLROS regulated mTOR signals in two manners. In the one hand, HAGLROS competitively sponged miR-100-5p to increase mTOR expression by antagonizing miR-100-5p-mediated mTOR mRNA inhibition. On the other hand, HAGLROS interacted with mTORC1 components to activate mTORC1 signaling pathway which was known to be an important negative signal of autophagy. Here activation of mTORC1 signaling pathway by HAGLROS inhibited autophagy, thereby promoted excessive proliferation and maintained the malignant phenotype of GC cells.

CONCLUSION: The present study demonstrates that HAGLROS overexpression contributes to GC development and poor prognosis and will be a target for GC therapy and further develop as a potential prognostic biomarker.

Citation Format: Jinfei Chen, Fen Yang. STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells contributes to the malignant progression of gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-395.
Abstract SY07-02: Epigenetic regulation of T-cell exhaustion: Implications for cancer immunotherapy
Authors: Youngblood Benjamin A. , Ghoniem Hazem , Moustaki Ardiana , Abdelsamed Hossam , Fan Yiping , Thomas Paul , Stewart Elizabeth , Federico Sara
Published: August 2018

Immune checkpoint blockade (ICB)-mediated rejuvenation of CD8 T-cell effector functions has emerged as one of the most promising frontiers for treating cancer and chronic infections. However, antigen-specific T cells that have experienced prolonged antigen exposure are often terminally differentiated, and have a limited capacity to mount an effector response during ICB treatment. Such exhaustion of effector potential is a major impediment of current T cell-based immunotherapy efforts. Using in vivo mouse models of tumor and chronic viral infection, we assessed the role of de novo epigenetic programming in establishing ICB-refractory exhausted T cells. We observed that genetic deletion of the de novo DNA methyltransferase, Dnmt3a, in T cells at the effector stage of an immune response to chronic lymphocytic choriomeningitis virus (LCMV) infection allowed antigen-specific T cells to remain highly functional despite expressing high levels of PD-1 and having prolonged exposure to antigen. Quite strikingly, PD-1 blockade treatment of chronically infected animals resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Whole-genome methylation profiling of WT and Dnmt3a-deficient LCMV-specific CD8 T cells identified de novo DNA methylation programs that are coupled to development of ICB-nonresponsive virus and tumor-specific T cells. Building upon these findings, we have identified de novo epigenetic programs acquired in human tumor-associated PD-1hi CD8 T cells. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key regulator in establishing ICB-refractory exhausted CD8 T cells and highlight epigenetic reprogramming of T cells as a novel approach to enhance T cell-based cancer therapies.

Citation Format: Benjamin A. Youngblood, Hazem Ghoniem, Ardiana Moustaki, Hossam Abdelsamed, Yiping Fan, Paul Thomas, Elizabeth Stewart, Sara Federico. Epigenetic regulation of T-cell exhaustion: Implications for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY07-02.
Abstract SY22-02: Metabolic vulnerability of PTEN mutant cancer
Authors: Parsons Ramon E.
Published: August 2018

Signaling pathways that act as drivers of cancer cell growth and proliferation activate metabolic pathways required to generate the building blocks required for cell doubling. Loss of PTEN activates PI3K, AKT and mTOR to stimulate glucose and glutamine flux to allow DNA replication. One of the metabolic pathways that is activated after deletion of both alleles of PTEN is the de novo pyrimidine synthesis pathway. PTEN null cells show evidence of increased DNA replication and low levels of damage at replication forks that can be rescued by providing supplementary uridine. Interruption of de novo pyrimidine synthesis with an inhibitor of dihydroorotate dehydrogenase (DHODH) increased DNA damage at replication forks of PTEN null cells that ultimately led to chromosome breaks detected in mitosis and cell death. This cellular vulnerability to inhibition of DHODH was rescued with uridine, demonstrating the specificity of the effect. Exploration of the mechanism that led to vulnerability of PTEN null cells demonstrated that it was due to defective ATR signaling at replication forks that was a consequence of activated AKT, which phosphorylate and attenuate TOPBP1 and CHK1. Complementation of PTEN null cells with mutants of TOPBP1 and CHK1 that cannot be phosphorylated by AKT rescued the cells from DNA damage and cell death due to inhibition of DHODH. Tumor cell lines lacking PTEN tended to be more sensitive to DHODH inhibition than PTEN wild type cells were. In conclusion, the attenuated ATR signaling and increased flux of nucleotides to newly synthesized DNA at replication forks makes PTEN mutant cells vulnerable to disruption of de novo pyrimidine synthesis.

Citation Format: Ramon E. Parsons. Metabolic vulnerability of PTEN mutant cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY22-02.
Abstract A03: Circulating CCL2 and CXCL8 chemokine levels and receptor interactions differ in African-Americans and Caucasians with breast cancer
Authors: Jenkins Brittany Denise , Hire Rupali , Martini Rachel N. , Davis Melissa B.
Published: August 2018

In 2013, the breast cancer mortality rate of African-American (AA) women in the U.S. was significantly higher than Caucasian women, even though incidence rates were about equal. Undoubtedly, access to health care and socioeconomic status could contribute to this health disparities gap, but inherent biologic differences may also be a contributing factor. The purpose of this study is to determine if differences in immunobiology, particularly the abundance of two proinflammatory chemokines, differs between AA and Caucasian women with breast cancer. Differences in these signaling molecules can affect the migration of immune cells, in addition to having a significant effect on cancer progression and tumor biology. We are also interested in the interactions between these chemokines and one of their receptors, ACKR1, which harbors ancestry-specific alleles. Generally, those of AA descent express less of this receptor on their tumor cells, producing a yet to be determined immune response when it comes to breast cancer.

Using plasma samples obtained from breast cancer patients pretreatment, in addition to plasma samples from control patients, we were able to determine any differences in chemokine protein levels from these participants using a Luminex immunoassay. Our molecules of interest, CCL2 (MCP-1) and CXCL8 (IL-8), were both chosen due to their ability to influence immune cell migration, in addition to angiogenesis and cancer cell progression. From our analysis, race-specific differences were observed for these particular chemokines between AA and Caucasians with breast cancer. When considering CCL2, it appears that AA had significantly less CCL2 present in circulation when compared to Caucasians. For CXCL8, the median concentration for AA cancer patients was much lower than that of the Caucasian group. The presence or absence of ACKR1 on the tumors of these patients also seems to play a role in circulating chemokine levels, with higher circulating levels being associated with those who have the receptor present on tumor cells. Overall, there appear to be biologic differences in immune response between AA and Caucasians with breast cancer, especially when considering CCL2 and CXCL8, and their interactions with ACKR1. This difference could account for variations in treatment responses, in addition to differences in breast cancer aggressiveness and progression seen within these two groups.

Citation Format: Brittany Denise Jenkins, Rupali Hire, Rachel N. Martini, Melissa B. Davis. Circulating CCL2 and CXCL8 chemokine levels and receptor interactions differ in African-Americans and Caucasians with breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A03.
Abstract A08: Epigenetic heterogeneity in triple-negative breast cancer
Authors: Maruyama Reo , Yang Liying , Kumegawa Kohei
Published: August 2018

Triple-negative breast cancer (TNBC) is defined by the absence of estrogen/progesterone receptor (ER/PgR) expression and HER2 amplification. Patients with TNBC have a higher rate of distant recurrence and a poorer prognosis than women with other breast cancer subtypes, since targeted therapy is not available for them. TNBC is a highly heterogeneous group of tumors and the underlying biology of these tumors should be substantially different. Stratification is essential for selection of appropriate therapy. We have previously identified key regulators that define basal-like (ER-/PgR-/HER2-) and luminal (ER+/PgR+ and/or HER2+) breast cancer cell phenotypes by using combined somatic cell genetic and integrative epigenomic approaches (Su et al., Cell Reports 2015). We found that common core epigenetic programs in luminal tumors are defined by FOXA1 super-enhancers; however, apart from displaying extinguished luminal features, the basal-like phenotype is not defined by a specific epigenetic state and displays a high degree of heterogeneity.

In this study, we aimed to identify a key regulator that can classify TNBCs into two groups, so that it could help improve our understanding of biology and explain the epigenetic heterogeneity in TNBC. We first reanalyzed our original multi-omics data of hetero-fusion cells in the previous study and also performed an integrated analysis of genomic, transcriptomic, and epigenomic data obtained from the TCGA project. We finally focused on a transcription factor (TF-X), whose function is currently unknown. TF-X is upregulated in a subset of TNBCs and the expression level of the gene is associated with poor prognosis in TNBC. Knockdown of TF-X decreased cell survival rate and changed cellular morphology of the cells. We are currently doing further functional analysis of TF-X. Our findings seem to imply that TF-X is one of the key regulators that govern cellular property of TNBC.

Note: This abstract was not presented at the conference.

Citation Format: Reo Maruyama, Liying Yang, Kohei Kumegawa. Epigenetic heterogeneity in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A08.
Abstract A11: Association of the main variants of the 8q24- rs13281615 and 2q35-rs13387042 with breast cancer risk in Khorasan population
Authors: Pasdar Alireza , Nesay Abolfazl , Tajbaksh Amir , Rivandi Mahdi , Javan Fahime Afzal , Moezzi Atefe , Toroghian Younes , Ghasemi Amirhossein , Soltanian Hossein , Salehi Mahta , Shandiz Fatemeh Homaei
Published: August 2018

Background: Since 2013, the incidence rate of breast cancer has remained as the first rank among female cancers worldwide. Breast cancer is a heterogeneous disease that has different environmental and genetic risk factors. In recent years, advances in molecular techniques resulted in the inaccurate characterization of molecular markers and therefore improvement of diagnostic and treatment methods. GWA studies play a key role in determining low-penetrance variants that influence the susceptibility of breast cancer. Studies have shown the strong association of two markers, rs10995190 and rs10822013, on chromosome 10q21.2, with breast cancer among different populations.

Considering genetic diversity among various populations and the fact that the association of these markers with the risk of developing breast cancer has not been investigated in our population, our study aimed to investigate if these markers are associated with increased risk of breast cancer in the Khorasan province population.

Methods: The current study was conducted on 400 women, including 185 breast cancer patients and 215 healthy controls. Genotyping was performed using ARMS_PCR method.

Results: There was a significant difference in rs10822013 genotype allele frequencies between patient and control groups (P<05). However, there were no significant differences in genotype and allele frequencies between patient and control group (P=0.41) for rs10995190.

Conclusion: Based on the results, rs10822013 may increase the risk of breast cancer in our population, which is in line with the other results in the Asian population.

Citation Format: Alireza Pasdar, Abolfazl Nesay, Amir Tajbaksh, Mahdi Rivandi, Fahime Afzal Javan, Atefe Moezzi, Younes Toroghyan, Amirhossein Ghasemi, Hossein Soltanian, Mahta Salehi, Fatemeh Homaei Shandiz. Association of the main variants of the 8q24- rs13281615 and 2q35-rs13387042 with breast cancer risk in Khorasan population [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A11.
Abstract A12: Characterization of the mitronic tsmiR miR6855-3p as a DNA damage repair regulator in the SLUG-high metastatic breast cancer cells
Authors: Ellison Marshall JD , Misra Smita , Chaudhuri Gautam
Published: August 2018

The precursor of the human microRNA hsa-miR-6855-3p is a 5-tailed mitronic pre-miRNA that is coded by intron #13 of the deubiquitinase USP20 gene. Our preliminary data suggest that the biosynthesis of this miRNA is upregulated by oxidative stress in the SLUG-high metastatic breast cancer cells, promoting DNA double-strand break repair (DSBR) through the indirect enhancement of BRCA2 gene expression. We found that miR-6855-3p favors BRCA2 biosynthesis through the enhancement of the level of an alternately translated form of the BRCA2 gene enhancer protein SPRDX5. We hypothesize that BRCA2 gene silencer acts as a scaffolding to co-recruit SLUG and SPRDX5 and promote SPRDX5-mediated oxidative inactivation of the BRCA2 negative regulator protein SLUG. Here, we present evidence that (a) the expression of miR-6855-3p is essential for the expression of BRCA2 in the SLUG-high metastatic breast cancer cells; (b) knockdown of miR-6855-3p affects DSBR in these cells; and (c) the miR-6855-3p knocked-down cells are susceptible to the synthetic lethality by the PARP inhibitors. Our study reveals a novel mechanism of regulation of BRCA2 gene expression in the aggressive breast cancer cells, offering an alternative strategy to chemically intervene in the progression of hard-to-cure, highly aggressive SLUG-high breast cancer. We propose that the inhibition of miR-6855-3p will induce BRCA2 deficiency in the BLBC cells, thus increasing their susceptibility to standard chemotherapy as well as to PARP inhibitors as has been described for other BRCA2-deficient cells. Supported in part by DOD-CDMRP IDEA Expansion Grant #BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and 1U54RR026140 to SM.

Note: This abstract was not presented at the conference.

Citation Format: Marshall JD Ellison, Smita Misra, Gautam Chaudhuri. Characterization of the mitronic tsmiR miR6855-3p as a DNA damage repair regulator in the SLUG-high metastatic breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A12.
Abstract A17: Ganoderma lucidum inhibits the DNA damage response in combination with carboplatin in breast cancer cells
Authors: Suárez-Arroyo Ivette , Martínez-Montemayor Michelle
Published: August 2018

Management of triple-negative breast cancer (TNBC) is challenging because of a lack of targeted therapy, aggressive behavior, and poor prognosis. Interest in using platinum agents, such as carboplatin, as a line of treatment emerged from data suggesting frequent DNA repair defects in TNBC. Carboplatin resistance may occur due to multiple factors including increased activation of DNA repair mechanisms. Studies show that Ganoderma lucidum extract (GLE) sensitizes cancer cells to different anticancer therapies. Thus, we aim to investigate the effects of GLE in combination with carboplatin and to elucidate its effects in the DDR signaling. To study whether GLE sensitizes breast cancer cells to carboplatin therapy, we treated SUM-149 and MDA-MB-231 triple-negative breast cancer cell lines with several concentrations of carboplatin, GLE, or both treatments simultaneously for 72h. Our results demonstrate that when carboplatin and GLE are used in combination, the IC50s decreased in both breast cancer cell lines in comparison with treatments alone. Moreover, our data revealed that GLE and/or the combination decrease the expression of 53BP1, p-CHK2, p-CHK1, p-ATR, p-ATM, and p-BRCA1 in SUM-149 and/or in MDA-MB-231 cells. In addition, the phosphorylation of the pro-apoptotic protein p53 was upregulated by the combination. Immunofluorescence studies on DNA repair proteins 53BP1 and phosphorylated γH2AX to detect DNA double-strand breaks suggest that the GLE and/or carboplatin combination impair DDR. Taken together, our results provide evidence of GLE’s potential in chemosensitizing TNBC cells to carboplatin therapy affecting the DDR. These results highlight GLE’s anti-breast cancer therapeutic potential. This work is supported by NIGMS SC3GM111171 (MMM), PRSTT SGRP 2016-2017 (MMM), NIMHD MD007583 (MMM), and Title-V-PPOHA P031M105050 and Title-V-Cooperative P031S130068 from the U.S. Dept. of Education.

Note: This abstract was not presented at the conference.

Citation Format: Ivette Suárez-Arroyo, Michelle Martínez-Montemayor. Ganoderma lucidum inhibits the DNA damage response in combination with carboplatin in breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A17.
Abstract A25: TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer
Authors: Byrd Tiara , Fousek Kristen , Pignata Antonella , Szot Christopher , Samaha Heba , Dobrolecki Lacey , Oo Htoo Zarni , Sorensen Poul , Ellis Matthew , Lewis Michael , Hegde Meenakshi , Fletcher Bradley , Croix Brad St. , Ahmed Nabil
Published: August 2018

Lacking marked expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), triple-negative breast cancer (TNBC) is a breast cancer subtype in desperate need of targeted therapy options. Tumor endothelial marker 8 (TEM8), initially identified as a tumor endothelium marker in colon cancer, has been shown to be upregulated in TNBC. To confirm this, we stained primary TNBC tissues for TEM8; in all cases TEM8 was expressed with no expression in normal breast tissue. TEM8 is expressed by TNBC cell lines as indicated by flow cytometry and Western blot. We thus engineered chimeric antigen receptor (CAR) T cells to specifically target TEM8 in TNBC. TEM8 CAR T cells distinctly recognized TEM8, secreted immunostimulatory cytokines, and killed TEM8-positive TNBC cells in vitro. In vivo, the adoptive transfer of TEM8 CAR T cells induced regression against orthotopic patient-derived xenograft (PDX) models, including the aggressive claudin-low TNBC PDX, WHIM12. Systemic administration of TEM8 CAR T cells also induced regression against a lung metastasis TNBC model. In all models, treatment with TEM8 CAR T cells resulted in a survival advantage in mice compared to controls. Hence, TEM8 may serve as an attractive targeted immunotherapy of TNBC.

Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Heba Samaha, Lacey Dobrolecki, Htoo Zarni Oo, Poul Sorensen, Matthew Ellis, Michael Lewis, Meenakshi Hegde, Bradley Fletcher, Brad St. Croix, Nabil Ahmed. TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A25.
Abstract A35: Characterizing the role of the nuclear coactivator AIB1 in triple-negative breast cancer
Authors: Saenz Francisco R. , Wellstein Anton , Riegel Anna T.
Published: August 2018

Individuals diagnosed with triple-negative breast cancer (TNBC), a highly invasive and frequently metastatic breast cancer subtype, do not benefit from targeted therapies. TNBC is a heterogenous disease and can be further subdivided by intrinsic molecular subtyping into basal-like 1 or 2 (BL-1, BL-2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptor (LAR). Amplified in Breast Cancer 1 (AIB1) is an oncogene that is frequently overexpressed in higher-grade and invasive breast cancers. AIB1 regulates the transcription of several genes through hormone or growth factor signaling. Many of these signaling pathways are involved in cellular growth, differentiation, migration, and metabolism—all of which become deregulated during oncogenesis. In this study, we silence AIB1 by shRNA and study the phenotypic effects in a panel of TNBC cell lines from different TNBC subtypes in vitro. We have established TNBC HCC1806 cell lines expressing low levels of AIB1 and have characterized their phenotype compared to scramble control cells. We observe significant reduction in AIB1 message and protein as detected by RT-qPCR and Western blot. shAIB1 TNBC cells have a significant reduced survival following the initial knockdown. However, serial passaging of surviving cells show differences in proliferation, especially in low serum conditions. These AIB1LOW cells have a significant increase in motility shown in chemotaxis experiments. However, we observe delayed adhesion of AIB1LOW cells when seeded onto plastic culture vessels. Furthermore, when grown onto Matrigel, AIB1LOW cells have a significant reduced tube formation capacity related to their delayed adhesion or reduced motility on substratum. Additionally, AIB1LOW cells have a differential expression of cell surface markers associated with cancer stem cells (i.e., CD24, CD44, CD133). Consistent with these findings, AIB1LOW cells generate tumorspheres more efficiently under serum free in suspension and when embedded in Matrigel. Moreover, AIB1LOW cells develop aggressive phenotypically different tumors in the cleared mammary fat pad of nude mice compared to controls. The AIB1LOW cells are significantly less sensitive to chemotherapy agents and may represent a resistant population that emerges from cancer stem cells following therapy. Interestingly, AIB1LOW cells have a different regulation of genes associated with hypoxia, glycolysis, and apoptosis and these changes may give guidance as to therapy following emergence of resistance in TNBC patients.

Citation Format: Francisco R. Saenz, Anton Wellstein, Anna T. Riegel. Characterizing the role of the nuclear coactivator AIB1 in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A35.
Abstract A37: Combined targeting of estrogen receptor alpha and nuclear transport pathways remodels metabolic pathways to induce apoptosis and overcome tamoxifen resistance
Authors: Cotul Eylem Kulkoyluoglu , Wrobel Kinga , Yosef Landesman , Madak-Erdogan Zeynep
Published: August 2018

Majority of breast cancer specific deaths occur in women with recurrent, ERα (+), metastatic tumors that are endocrine therapy resistant. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and XPO1, a nuclear transport protein in overcoming endocrine resistance. Using Cignalfinder pathway profiling, Seahorse metabolic profiling, and GC/MS whole metabolite profiling, we found that combination of 4-OH-Tam and selinexor (SXR), an XPO1 antagonist being evaluated in multiple later-stage clinical trials in patients with relapsed and /or refractory hematologic and solid tumor malignancies, inhibited Akt phosphorylation by changing the localization of the kinase. Since we observed dramatic changes in Akt activity, we hypothesized that glucose utilization pathways and consequently metabolic profile of breast cancer cells would change in the presence of 4-OH-Tam and SXR. These cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source. In order to examine metabolites that might result in the observed phenotype, we performed whole metabolite profiling and identified proline metabolic pathways to be upregulated when cells were treated with SXR+4-OH-Tam. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways, increases demand on mitochondria, and causes increased production of ROS that would eventually lead to apoptosis. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine-resistant tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences.

Citation Format: Eylem Kulkoyluoglu Cotul, Kinga Wrobel, Landesman Yosef, Zeynep Madak-Erdogan. Combined targeting of estrogen receptor alpha and nuclear transport pathways remodels metabolic pathways to induce apoptosis and overcome tamoxifen resistance [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A37.
Abstract A43: Increased cyclin A expression is associated with antiprogestin resistance and progesterone receptor isoforms ratio in experimental models of breast cancer
Authors: Ayre Marina , Bilinski Melina E. , Jacobsen Britta M. , Lanari Claudia , Fabris Victoria T.
Published: August 2018

Deregulation of cyclin A expression has been associated with prognosis and tamoxifen responsiveness in breast cancer. We have previously reported an overexpression of cyclin A in two antiprogestin-resistant tumors derived from a hormone-dependent mammary carcinoma induced by medroxyprogesterone acetate (MPA). MPA-induced tumors show high levels of estrogen (ER) and progesterone receptors (PR) and transit through different stages of hormone dependency. Taking into account that antiprogestin-sensitive tumors show higher levels of PR isoform A (PRA) than isoform B (PRB), and that the opposite ratio is observed in the antiprogestin-resistant tumors, we decided to examine the expression of cyclin A in other experimental models containing different PRA/PRB ratios. An increase in cyclin A expression was observed by Western blot (WB) in the acquired antiprogestin-resistant variant C7-2-HIR and in the constitutive resistant C7-HI tumor (1.7 fold and 1.5 fold, respectively) compared to the antiprogestin-responsive tumor C7-2-HI (p<0.05). The immunohistochemical analysis of paraffin sections of the tumors confirmed these findings, showing a higher percentage of cyclin A positive nuclei in the antiprogestin-resistant tumor C7-HI, versus the responsive tumor C7-2-HI (p<0.01), suggesting an association of cyclin A expression with endocrine resistance in the murine breast cancer model. Next we evaluated the expression of cyclin A in T47D human breast cancer cells that express both PR isoforms and in T47D-YA or T47D-YB cells that express either PRA or PRB, respectively. The WB analysis of the nuclear fraction of cell lysates showed an increase in cyclin A expression in the T47D-YB cells (1.5 fold), compared to T47D and T47D-YA cells (all the cells were serum starved; p<0.01). Immunofluorescence for cyclin A using cultured breast cancer cells revealed a higher percentage of cyclin A positive nuclei in T47D-YB cells compared to T47D cells (p<0.001) or T47D-YA (p<0.01), respectively. Similarly, T47D-YB xenografts showed an increase of cyclin A expression by WB (1.47 fold) compared to T47D and T47D-YA tumors (p<0.05). These results suggest an association between cyclin A expression, the PR isoform ratio, and antiprogestin resistance. Cyclin A may participate in the ligand independent activation of PR in the antiprogestin-resistant variants of these experimental models of breast cancer.

Citation Format: Marina Ayre, Melina E. Bilinski, Britta M. Jacobsen, Claudia Lanari, Victoria T. Fabris. Increased cyclin A expression is associated with antiprogestin resistance and progesterone receptor isoforms ratio in experimental models of breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A43.
Abstract A50: Modeling and targeting of oncogenic proteotoxic stress in drug-resistant breast cancer
Authors: Singh Navneet , Modur Vishnu , Muhammad Belal , Komurov Kakajan
Published: August 2018

ERBB2/HER2-positive breast cancers are associated with poor prognosis, and resistance to HER2-targeted therapy (trastuzumab and lapatinib) presents a significant clinical hurdle. There are no treatment options for metastatic HER2+ BCs that have progressed on HER2-targeted therapy. At present, most of the research on therapeutic strategies in therapy-refractory Her2+ BCs focuses on resensitizing tumor cells to HER2-targeting agents. We have shown that targeting of nononcogenic vulnerabilities, specifically adaptive proteotoxic stress response, is a promising therapeutic approach in this subset of BCs (Singh N. et al, HER2-mTOR signaling-driven breast cancer cells require ER-associated degradation to survive. Science Signaling 2015). We showed that Her2+ BCs have specific dependence on the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to suppress the hyperactive Her2/mTOR pathway-driven proteotoxic stress. Here, we explore the kinetics of protein homeostatic changes during the course of Her2-targeted drug therapy, and identify optimal therapeutic windows for the targeting of ERAD in the therapy-refractory Her2+ BCs.

Using an integrated systems approach, we have modeled the dynamics of signaling and protein homeostasis changes in Her2+ cells during remission, resistance, and relapse phases of Her2-targeted therapy. We found that the resistance and relapse phases of anti-Her2 drug (lapatinib and trastuzumab) treatment are associated with Her2-independent acquisition of mTOR signaling, as previously reported. However, these signaling pathway changes also resulted in heightened proteotoxic burden on the ER due to increased protein synthesis rates, which was further exacerbated upon the release of cells from anti-Her2 treatment. Accordingly, cells in the relapse phase, and even more so upon further drug withdrawal, showed significantly heightened sensitivity to the inhibition of ERAD. This study reveals the kinetics of protein homeostasis associated with the signaling pathway changes during acquired resistance to anti-Her2 therapy, and suggests optimal therapeutic windows for the targeting of ERAD in therapy-refractory Her2+ BCs.

Citation Format: Navneet Singh, Vishnu Modur, Belal Muhammad, Kakajan Komurov. Modeling and targeting of oncogenic proteotoxic stress in drug-resistant breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A50.
Abstract A61: BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area blocks luminal epithelial differentiation
Authors: Chiang Huai-Chin , Zhang Xiaowen , Hu Yanfen , Li Rong
Published: August 2018

Most BRCA1-associated breast tumors are basal-like, yet originate from luminal progenitor cells. BRCA1 is best known for its functions in double-strand break (DSB) repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions of BRCA1 fully explains the cell lineage-specific increase in breast tumor development. By sorting and profiling lineage-specific cells from precancerous human breast tissue, we found that BRCA1 mutation carriers tend to have a luminal cell-specific accumulation of R-loop, a transcriptional byproduct of DNA-RNA hybrid structure important for transcriptional regulation and genomic instability. R-loops accumulate preferentially at enhancers and transcription start sites (TSS) of luminal fate-related genes. Using CRISPR-Cas9 system, we have deleted an ERα enhancer area with BRCA1-associated R-loops accumulation in MCF7 cells. The deletion of this noncoding putative ERα enhancer area reduced the gene expression level of the neighboring genes of the deleted region, including ERα, RMND1, and CCDC170. In addition, compare to the reduced expression level of ERα, RMND1, and CCDC170 in BRCA1 knockdown of the parental MCF7, knocking down BRCA1 in these deletion clones showed less reduction of these genes. On the other hand, overexpression of RNASEH1, an enzyme that specifically removes R-loop, had little effect in these genes after BRCA1 knockdown. These results indicate BRCA1-associated R-loop accumulation at least partially affects luminal gene transcription. To investigate whether BRCA1-assocaited R-loop accumulation directly contributes to luminal differentiation blockage, we overexpressed RNASEH1 in breast epithelial cells freshly isolated from BRCA1 mutation carriers; flow cytometry comparing different breast lineage revealed a luminal population shift toward more mature luminal. All in all, the results imply that BRCA1 promotes luminal epithelial differentiation by alleviating R-loop accumulation at enhancer of ERα. Aberration in luminal transcription could contribute to lineage-specific BRCA1-associated tumorigenesis.

Citation Format: Huai-Chin Chiang, Xiaowen Zhang, Yanfen Hu, Rong Li. BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area blocks luminal epithelial differentiation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A61.
Abstract B26: Thermally abused frying oil enhances metastatic progression in vivo and in vitro: A link to elevated fumarate levels
Authors: Hughes Jennifer R. , Joyce Kaitlyn M. , Cam Anthony , Helferich William G. , Madak-Erdogan Zeynep
Published: August 2018

For the past four decades, dietary fat intake has composed a significant portion of the typical Western diet, at 33-35% of total kilocalories. Approximately 8% of this dietary fat is from consumption of deep fat fried foods. Oil has been demonstrated to be absorbed by food during frying and contributes between 8 to 40% of the food’s final weight. As oils are used and reused for frying, complex chemical reactions take place creating deleterious polar compounds (e.g., acrylamide, sterol derivatives, or other reactive oxidative species.) Although these compounds are associated with health risks, i.e., heart disease, diabetes mellitus, and cancer, there is no regulation in the United States for upper limits of polar material in frying oil. This raises an important question as to whether thermally abused fryer oil (TAFO) can alter metastatic breast cancer progression. We initially conducted an in vivo study in which a modest amount of dietary fat, 25% of kilocalories, came from fresh fryer oil or TAFO. Ovariectomized BALB/C mice fed the diet containing TAFO for 16 weeks and inoculated with luciferase-expressing 4T1 murine mammary cancer cells had significantly more metastasis to lungs than control-fed animals (p < 0.05, n=21). Serum metabolic analysis in these animals revealed significantly elevated levels of fumarate, a known onco-metabolite, in TAFO-fed compared with control-fed animals (p < 0.05, n=9). To confirm that fumarate was mechanistically involved in metastatic progression, we conducted in vitro studies. 4T1 cells exposed to 500 nM to 10 µM monomethyl fumarate (MMF), a cell-permeable form of fumarate, trended toward an increase in the healing rate over 24 hours in a wound healing (scratch) assay compared to control (n =2). This preliminary evidence supports the hypothesis that fumarate accumulation creates a protumorigenic and metastatic environment and needs confirmation in subsequent investigation. This information can help elucidate mechanistic details of the effects of TAFO on breast cancer metastasis. Further work will assess rates of 4T1 cellular proliferation and invasion induced by MMF. In summary, dietary intake of TAFO increased metastasis of tumor cells to lungs, and dysregulation of the metabolite fumarate may be mechanistically involved.

Citation Format: Jennifer R. Hughes, Kaitlyn M. Joyce, Anthony Cam, William G. Helferich, Zeynep Madak-Erdogan. Thermally abused frying oil enhances metastatic progression in vivo and in vitro: A link to elevated fumarate levels [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B26.
Abstract B35: Palbociclib treatment activates FAK and use of palbociclib in combination with the FAK inhibitor PF-562,271 enhances antitumor activity in ER-positive breast cancer cells
Authors: Howe Grant , Allen Victoria , Addison Christina L.
Published: August 2018

Cell survival and proliferation is tightly regulated by a number of interacting coordinated signaling proteins including the cyclin-dependent kinases (CDK). Palbociclib, a CDK4/6 inhibitor, has recently been shown to be very effective in the treatment of estrogen receptor (ER) positive breast cancer; however, many patients develop an acquired resistance through as yet incompletely characterized mechanisms. One recently proposed mechanism is compensation by CDK2 (Herrera-Abreu et al., Can Res 2016;76:2301). Focal adhesion kinase (FAK) is a tyrosine kinase that plays a central role in signaling pathway crosstalk and cell survival and invasion. FAK has been previously shown to activate CDK2 in hepatocytes (Flinder et al., J Cell Phys 2013;228:1304), and given its important role in mediating cell survival, we were interested in determining whether FAK-mediated upregulation of CDK2 occurs in palbociclib-treated breast cancer cells, thereby limiting its efficacy. ER-positive MCF7, MDA-MB-134VI, and T47D breast cancer cell lines were thus treated with increasing doses of palbociclib. We observed dose-dependent increases in phospho-FAK Y397, a marker of the active kinase, following palbociclib treatment. We thus used PF-562,271, a selective FAK tyrosine kinase inhibitor, in combination with palbociclib and, not surprisingly, observed an enhanced ability to inhibit cell viability as compared to use of either drug alone. This was concomitant with observed increases in the CDK4/6 inhibitor p27, and with decreases in phospho-RB, which are indicative of reduced CDK2 activity, following treatment with PF-562,271. Our results highlight an unexpected side effect of palbociclib treatment, namely FAK activation, which could potentially contribute to resistance due to activation of CDK2 and inhibition of p27. The mechanism by which palbociclib activates FAK is currently under investigation.

Citation Format: Grant Howe, Victoria Allen, Christina L. Addison. Palbociclib treatment activates FAK and use of palbociclib in combination with the FAK inhibitor PF-562,271 enhances antitumor activity in ER-positive breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B35.
Abstract B45: Studying the interactome of breast cancer: The cancer cell map initiative
Authors: Kim Minkyu , Kim Kyumin , Soucheray Margaret , Swaney Danielle , Krogan Nevan
Published: August 2018

Recent progress in genome sequencing has revealed numerous mutations in cancer genomes, but how many of these alterations result in changes in normal cellular processes is poorly understood. In addition, the biologic functions of the majority of genes (e.g., BRCA1/2) with cancer-associated mutations have not been fully characterized, despite their expression and/or activity being highly correlated with cancer. Through years of study, it is becoming clear that cancer is a disease that arises not only because of defects in individual genes and proteins, but also because of the action of hallmark cellular processes and biologic pathways. Therefore, what is urgently needed is to put the cancer genomic information into biologic context by mapping mutated genes onto the complexes and pathways in which they function. The goal of this study is to uncover the comprehensive protein-protein interaction networks and pathways in various breast cancer subtypes to better understand how mutated cancer genes and genomes hijack and rewire pathways and complexes during the course of breast tumorigenesis. Here we catalog protein-protein interactions for more than 40 genes recurrently mutated in breast cancer, using affinity purification and mass spectrometry (AP-MS). Our interaction network reveals subtype and mutation-specific protein-protein interactions, most of which are not previously reported. We anticipate the breast cancer interactome study will uncover many previously unidentified aberrant pathways and protein complexes uniquely operating in breast cancer cells, and thus pinpoint proteins central in these pathways and complexes that may potentially serve as distinct biomarkers or therapeutic targets for tumors having the same or similar subtypes and/or genomic mutations.

Citation Format: Minkyu Kim, Kyumin Kim, Margaret Soucheray, Danielle Swaney, Nevan Krogan. Studying the interactome of breast cancer: The cancer cell map initiative [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B45.