Breast Cancer Cell Line Models Research Articles

Abstract 2935: Systematic drug testing and RNA-sequencing of tamoxifen resistant breast cancer cell lines

Abstract Tamoxifen, as a standard treatment of estrogen receptor (ER)-positive breast cancer, reduces breast cancer mortality by 31%. In 50% of advanced ER-positive cancers, however, de novo resistance exists and in 40% of patients with initial response acquired resistance frequently evolves. In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new opportunities to treat patients, we created seven tamoxifen-resistant breast cancer cell line models that represent the luminal A subtype (MCF-7, T-47D, ZR-75-1), which expresses ER, and the luminal B subtype (BT-474), which additionally expresses the HER2 oncoprotein. We then performed drug sensitivity and resistance testing (DSRT), exome-sequencing and network analysis on all these cancer cell lines to determine the molecular and drug response profiles specific to tamoxifen resistance. As the cells became tamoxifen-resistant, we observed increasing sensitivity towards drugs like ERK1/2-, proteasome- and BCL-family inhibitors, but each of the isogenic cell line pairs had its distinct genomic and drug response profile. We then studied the molecular profiles of the 7 drug-resistant variants by RNA-sequencing in comparison to their 4 isogenic parental cells. We could not detect any common significantly differentially expressed genes (more than 2 fold change) across all the cell lines. However, the cell lines could be grouped into two different categories: The ones with low amount of differentially expressed genes, < 800 genes in the BT-474 and ZR-75-1 cell line pairs (with a 1,2% overlap), and the ones with high amount of differentially expressed genes, >1800 genes in T-47D and MCF-7 (with 7,3% overlap). Further analysis with the Ingenuity pathway analysis tool revealed an involvement of “Fatty Acid Activation” as well as “Stearate Biosynthesis I” in this group. Additionally, we discovered that genes involved in iron metabolism (TFRC, IREB2 and FTL) or iron-regulated genes like CP and NDRG1 are deregulated. These genes and pathways thereby provide avenues to identify new drug vulnerabilities for the tamoxifen resistant cancer cells, which we are now investigating at gene and protein level e.g. by image-based phenotyping. In summary, by combining drug testing data with the RNA-sequencing results, we hope to provide a number of potential drugs as well as matching biomarkers for planning clinical trials for patients with tamoxifen-resistant breast cancer. Citation Format: Susanne Hultsch, Sara Kangaspeska, Matti Kankainen, Vilja Pietiäinen, Olli Kallioniemi. Systematic drug testing and RNA-sequencing of tamoxifen resistant breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2935.

Abstract LB-200: Integrating dynamic 18F-FDG PET-CT, tumor metabolomics and functional genomics to understand metformin's pharmacodynamic effects in breast cancer: results of a phase 0 clinical trial

Abstract There is growing interest in the anticancer effect of the diabetes drug, metformin, and over 100 clinical trials are underway worldwide. However it is still not determined as to whether metformin has significant direct effects on cancer cells or solely indirect effects via modulation of host metabolism. We recruited 41 non-diabetic patients with primary breast cancer to a neoadjuvant window trial. Patients received an escalating dose of metformin (500mg up to 1500mg) for 2 weeks with pre- and post-metformin pharmacodynamic assessments including, dynamic 18F-FDG PET-CT scans, serum metabolic markers, and tumour biopsies for whole transcriptome RNASeq, tumour metabolomics and immunohistochemistry. Assessment of tumour FDG kinetics using a classic 2-tissue compartment model with three rate constants showed a 1.3 fold change (FC) post-metformin in the composite 18F-FDG flux constant, Kflux (p = 0.041, on paired t-test). There was a non-statistically significant 1.1 fold increase in the variable, metabolic rate of glucose utilization (MRglu) (p = 0.141). Mass spectrometry analysis revealed a decrease in intra-tumoral levels of the short-chain acyl-carnitines, propionylcarnitine (FC -0.50, p = 0.039) and acetylycarnitine (FC -0.40, p = 0.046) consistent with inhibition of fatty acid oxidation (wilcoxon rank test). This effect on fatty acid oxidation was validated in pre-clinical in vitro and in vivo breast cancer cell line models. There was a statistically significant positive correlation between the actual difference in Kflux and intratumoral levels of acetylcarnitine (p = 0.012, Spearman's rank test). Small but consistent falls in the levels of serum glucose (p = 0.032), c-peptide (p = 0.001), insulin (p = 0.005), IGF1 (p = 0.048) and IGF2 (p = 0.040) were observed following metformin treatment (paired t-test) but there was no correlation with change in Kflux or short chain acyl-carnitine levels. Pathway annotation analysis using GeneCodis revealed several pathways associated with mitochondrial and fatty acid metabolism that were upregulated. Immunohistochemical intratumoral nuclear expression of pAMPK increased 1.5 fold (p = 0.037, paired t-test) but this did not correlate with change in Kflux or levels of short-chain acyl-carnitines. Peak serum metformin levels correlated with intratumoral metformin levels (p = 0.012, Spearman's rank test) but did not correlate with change in Kflux or short-chain acyl-carnitine levels. Our data shows that metformin treatment alters FDG kinetics, inhibits fatty acid oxidation and leads to altered gene expression in the mitochondrial (nuclear encoded) transcriptome but that these effects do not correlate with changes in host metabolism. This data provides strong evidence that metformin has a direct effect on breast cancer metabolism at clinical doses. Citation Format: Simon Lord, Daniel Liu, Syed Haider, Edoardo Gaude, Eugene Teoh, Patel Neel, Qifeng Zhang, Fergus Gleeson, Michael Wakelam, Christian Frezza, Francesca Buffa, Fenwick John, Adrian Harris. Integrating dynamic 18F-FDG PET-CT, tumor metabolomics and functional genomics to understand metformin's pharmacodynamic effects in breast cancer: results of a phase 0 clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-200.

Abstract 3843: Design of effective combination therapies for high-grade serous ovarian cancer using patient-derived xenograft models

Abstract Despite advances in understanding the genetics and molecular biology of high grade serous ovarian cancer, there have not been any significant improvements in the outcome of patients treated with approved therapies to date. The overall aim of our studies is to identify synergistic drug combinations for the treatment of high-grade serous ovarian (HGS-Ov) cancer and the biomarkers that predict sensitivity for future translation in clinical trials. In particular, this study focuses on the vulnerabilities of patient-derived ovarian cancer cells to inhibition of anti-apoptotic proteins and the design of novel combination therapies to overcome drug resistance and improve the response of selected patients to a particular set of targeted therapies. Previous studies from our lab provided evidence that inhibition of BCL-2/XL can significantly enhance the sensitivity of tumor cells to targeted therapies in ovarian and breast cancer cell line models (Muranen T et al., Cancer Cell 2012). In this study, we evaluated the efficacy of this combination treatment in ascites cells derived from 15 high-grade serous patient-derived ovarian cancer xenografts (PDXs). We found that inhibitors of PI3K/mTOR (GNE-493) and BCL-2/XL (ABT-737) act synergistically ex vivo, with an ∼15 fold variability among the 15 patient-derived samples. A large scale in vivo experiment is ongoing to evaluate the efficacy of this combined treatment in six PDX models. To identify biomarkers that predict drug sensitivity we performed proteomic Reverse Phase Protein Array (RPPA) and immunoblot analyses. We found that baseline levels of the pro-apoptotic protein BIM correlate with sensitivity to BCL-2/XL inhibition. This motivated us to examine the basis for low BIM expression. In three of the least sensitive PDX models, we found that low levels of BIM correlated with ERK activation based on increased ERK phosphorylation and that inhibition of MEK by PD-0325901 in these models caused upregulation of BIM, as predicted from previous published reports which demonstrated that phosphorylation of BIM by ERK causes its degradation (Ley R, et al. J Biol Chem. 2003). We then examined the sensitivity of these PDX models to ABT-737 and PD-0325901, MEK162, or CI-1040 ex vivo and observed strong synergy resulting in reduction of cell viability and increased cell death. These results suggest that low BIM/high phosphoERK is a potential biomarker for sensitivity of ovarian tumors to combined MEK and BCL-2/XL inhibition. We now plan to examine the efficacy of drug combinations that target MEK and BCL-2/XL in these PDX models in vivo. Our studies promise to lead to the identification of new drug combination therapies for HGS-Ov cancer treatment and predictive biomarkers to stratify patients that can benefit from these targeted therapies Citation Format: Claudia Iavarone, Ioannis Zervantonakis, Hsing-Yu Chen, Sangeetha S. Palakurthi, Joyce F. Liu, Ursula Matulonis, Ronny Drapkin, Gordon Mills, Joel Leverson, Deepak Sampath, Joan Brugge. Design of effective combination therapies for high-grade serous ovarian cancer using patient-derived xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3843.

Abstract 178: PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer

Abstract Triple-Negative Breast Cancers (TNBCs) are aggressive, associated with poor prognosis and lack targeted therapies, mainly due to the absence of suitable molecular targets. The genomic region 6p21-p25 is recurrently amplified in TNBCs and encompasses the PIM1 oncogene. This study interrogated genomic breast cancer datasets and identified gene copy number-driven increase of PIM1 expression in TNBC. To understand the role of PIM1 in malignant phenotypes of TNBCs, functional studies were carried out in breast cancer and non-malignant mammary epithelial cell line models. RNA interference and rescue-of-function experiments revealed addiction to PIM1 kinase for cell population growth and apoptosis protection in TNBC cells, absent in non-malignant cells. In cells sensitive to PIM1 knockdown, we observed a subsequent reduction of the expression of the anti-apoptotic proteins BCL2 and MCL1. Moreover, exogenous expression of BCL2 prevented apoptosis caused by PIM1 knockdown. BH3-profiling analysis further confirmed that PIM1 blocks apoptosis elicited through the mitochondrial pathway in TNBC cell lines. The activity of PIM1 in other cancers has been closely linked to the regulation of c-MYC, an “un-targetable” oncogene that drives malignancy and that is frequently amplified and highly expressed in primary TNBCs and post-chemotherapy residual disease. Importantly, we found that PIM1 expression associates with several MYC-transcriptional signatures in TNBCs and mediates phosphorylation of c-MYC Ser62 and Histone-H3 Ser10, key targets for MYC-driven transcription. Exogenous expression of MYC rescued the phenotypes caused by PIM1 knockdown, providing mechanistic insights for the observed addiction. Finally, the therapeutic potential of targeting PIM1 was assessed using AZD1208, a small molecule inhibitor of the PIM kinase family. AZD1208 inhibited growth and sensitized TNBC derived cell lines, cell line xenografts and PDXs to chemotherapy by increasing apoptosis. We therefore identify PIM1 as a driver of malignancy in TNBC, illustrating relationships with MYC-activation and regulation of anti-apoptotic BCL2 and MCL1 proteins. PIM1 inhibitors could provide a potential therapeutic to abrogate chemotherapy resistance in TNBCs. Citation Format: Fara Braso-Maristany, Simone Filosto, Steven Catchpole, Rebecca Marlow, Jelmar Quist, Erika Francesch Domenech, Gianmaria Liccardi, Leila Zakka, Violeta Serra, Albert Gris, Maggie Cheang, Nirmesh Patel, Anna Perdrix Rosell, Patrycja Gazinska, Elodie Noel, Jonathan Watkins, Pierfrancesco Marra, Anita Grigoriadis, Andrew Tutt. PIM1, a novel target in chemotherapy-resistant triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 178.

Abstract 453: MEK activation is associated with a molecular subgroup in high grade serous ovarian cancer

Abstract Introduction We have previously defined 3 molecular subgroups of High grade serous ovarian cancer (HGSOC), ‘Angio’, ‘Immune’ and ‘Angio_immune’ subgroups using gene expression data from 265 FFPE HGSOC samples obtained from treatment naive patients and who were subsequently treated with platinum-based standard of care (SoC) chemotherapy (carboplatin +/- paclitaxel) (Gourley, et al. J Clin Oncol 32:5s, 2014). Patients within these 3 molecular subgroups respond differently to SOC treatment. The immune subgroup has the best outcome compared to the Angio and Angio_immune subgroups (HR of 0.63 and 0.66 respectively on multivariate analysis). Since it has previously been shown that the MAPK pathway is an important mediator of cisplatin resistance in ovarian cancer, we wanted to investigate if the MAPK pathway was associated with one of the poor prognosis subgroups. Methods A gene signature that could detect each of the subgroups Angio, Immune and Angio_immune was generated from the clinical samples. Data from the Cancer Genome Atlas (TCGA) Project was used to test for correlation between each subgroup and phospho-MEK as measured by Reverse Phase Proteomic Array (RPPA). Sensitivity to the MEK inhibitor trametinib (GSK1120212) and cisplatin was determined by 10-day colony formation assay. Results We found a statistically significant association between the Angio_immune subgroup signature and Phospho-MEK (serine 217/221) expression (p = 0.047) indicating activation of the MAPK pathway in this subgroup. Additionally we have demonstrated that the Angio_immune subgroup signature is suppressed by MEK inhibition (p = 0.0055) and elevated by KRAS, NRAS and MEK1 overexpression in cell line models (0.0072, 0.0004 and <0.0001). These effects were specific to the Angio_immune subgroup signature as there was no association with the Angio or immune subgroup signatures. Additionally the Angio_immune gene signature could predict resistance to cisplatin and sensitivity to MEK inhibitors in a panel of breast and ovarian cancer cell line models (p = 0.0017 and p = 0.0091). Acquired resistance to cisplatin in cell line models was also associated with MEK activation and an elevated Angio_immune gene signature score, which could be reversed by a MEK inhibitor. Conclusion We have identified a molecular subgroup in HGSOC that is associated with MAPK signalling. A gene signature to detect this subgroup from formalin fixed paraffin embedded samples has been developed and predicts sensitivity to MEK inhibitors in pre-clinical model systems. Further work aims to validate the signature in clinical samples from patients treated with a MEK inhibitor. Citation Format: Nuala McCabe, Charlie Gourley, Andrena McGavigan, Caroline O. Michie, Niamh McGivern, Michael Churchman, Eamonn J. O’Brien, Laura Hill, Timothy S. Davison, Alistair Williams, Glenn McCluggage, Karen E. Keating, Denis P. Harkin, Richard D. Kennedy. MEK activation is associated with a molecular subgroup in high grade serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 453.

Janus kinases and Src family kinases in the regulation of EGF-induced vimentin expression in MDA-MB-468 breast cancer cells

Epithelial-mesenchymal transition (EMT) is an important process associated with the metastasis of breast cancer cells. Members of the Janus kinases (JAKs) and Src family kinases (SFKs) are implicated in the regulation of an invasive phenotype in various cancer cell types. Using the pharmacological inhibitors JAK Inhibitor I (a pan-JAK inhibitor) and PP2 we investigated the role of the JAKs and SFKs, respectively, in the regulation of EMT markers in the MDA-MB-468 breast cancer cell line model of epidermal growth factor (EGF)-induced EMT. We identified selective inhibition of EGF induction of the mesenchymal marker vimentin by PP2 and JAK Inhibitor I. The effect of JAK Inhibitor I on vimentin protein induction occurred at a concentration lower than that required to significantly inhibit EGF-mediated signal transducer and activator of transcription 3 (STAT3)-phosphorylation, suggesting involvement of a STAT3-independent mechanism of EGF-induced vimentin regulation by JAKs. Despite our identification of a role for the JAK family in EGF-induced vimentin protein expression, siRNA-mediated silencing of each member of the JAK family was unable to phenocopy pharmacological inhibition, indicating potential redundancy among the JAK family members in this pathway. While SFKs and JAKs do not represent global regulators of the EMT phenotype, our findings have identified a role for members of these signaling pathways in the regulation of EGF-induced vimentin expression in the MDA-MB-468 breast cancer cell line.

The CYP17A1 inhibitor abiraterone exhibits estrogen receptor agonist activity in breast cancer.

Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone's ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.

Abstract P4-07-07: Evaluating Rad51/geminin protein expression as an indicator of homologous recombination deficiency in breast cancer models

Abstract Background: Homologous recombination deficiency (HRD) in cancer cells can occur due to mutations (germline or sporadic), methylation or other epigenetic causes. HRD leads to a defect in the conservative, error-free DNA repair mechanism and is associated with enhanced susceptibility to DNA targeting chemotherapy. Currently functional HRD assays are not broadly available for clinical use. Many of the HRD assays used in the experimental setting require fresh frozen tissue for optimal results, or require specialized expertise to interpret the results. We evaluated an immunohistochemical (IHC) assay using formalin fixed paraffin embedded (FFPE) tissue to measure protein expression of Rad51 and geminin, a cell proliferation marker, to assess HRD in breast cancer cell line models and clinical breast cancer samples. We hypothesize that Rad51, which is involved in the later stages of HR, can serve as a functional marker of HRD. Methods: The MCF-7 human breast cancer cell line was used as a model with intact HR. Western blotting of total cell lysates from cells grown in culture was performed to confirm HR response following treatment with DNA damaging chemotherapeutic agents, cisplatin and doxorubicin. Paclitaxel, a microtubule targeting agent, was used as a negative control. Mice with MCF-7 xenograft tumors were also treated with cisplatin, or doxorubicin at two dose levels (low and high) and various time points post treatment to assess the dose and time response to HR markers. Tumors from mice treated with paclitaxel were used as a negative control. Xenograft tumors were fixed and analyzed by IHC using an antibody specific for total Rad51 and geminin expression. DNA damage was also assessed in a portion of the tumor using a pulse gel electrophoresis assay. We also analyzed FFPE breast cancer clinical samples from patients with BRCA1 mutations for Rad51 and geminin expression. Results: In MCF-7 grown in vitro, total Rad51 was elevated as soon as 4 hours following exposure to doxorubicin and cisplatin, but not in response to paclitaxel treatment. In xenograft tumors, baseline Rad51 and geminin expression were relatively high illustrating proficient HR in an actively proliferating tumor model. Rad51 expression increased post treatment with cisplatin and doxorubicin as early as 6hrs and peaked at 16-24hrs. Geminin expression correlated well with expression of Rad51 at baseline and in time response to treatment. Pulse gel electrophoresis in paired tumor samples confirmed DNA damage was occurring compared to vehicle control treated tumors. However, this technique did not show a strong dose or time response. Five breast tumors from patients with known BRCA1 mutations were stained for Rad51 and geminin expression. High geminin expression and low Rad51 expression was noted in the majority of these tumors. Conclusions: An IHC assay using FFPE tissue to measure Rad51/geminin is a promising method to assess HRD in breast cancer. Further analytical and clinical validation of this approach is ongoing. Citation Format: Chalasani P, Nagy D, Livingston RB, Weterings E, Nagle R, Singh S, Barnes M, Grogan T, Ridder R, Baker AF, Kandavel S. Evaluating Rad51/geminin protein expression as an indicator of homologous recombination deficiency in breast cancer models. [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 P4-07-07.

Squalene epoxidase is a bona fide oncogene by amplification with clinical relevance in breast cancer.

SQLE encodes squalene epoxidase, a key enzyme in cholesterol synthesis. SQLE has sporadically been reported among copy-number driven transcripts in multi-omics cancer projects. Yet, its functional relevance has never been subjected to systematic analyses. Here, we assessed the correlation of SQLE copy number (CN) and gene expression (GE) across multiple cancer types, focusing on the clinico-pathological associations in breast cancer (BC). We then investigated whether any biological effect of SQLE inhibition could be observed in BC cell line models. Breast, ovarian, and colorectal cancers showed the highest CN driven GE among 8,783 cases from 22 cancer types, with BC presenting the strongest one. SQLE overexpression was more prevalent in aggressive BC, and was an independent prognostic factor of unfavorable outcome. Through SQLE pharmacological inhibition and silencing in a panel of BC cell lines portraying the diversity of SQLE CN and GE, we demonstrated that SQLE inhibition resulted in a copy-dosage correlated decrease in cell viability, and in a noticeable increase in replication time, only in lines with detectable SQLE transcript. Altogether, our results pinpoint SQLE as a bona fide metabolic oncogene by amplification, and as a therapeutic target in BC. These findings could have implications in other cancer types.

Abstract B48: Design of effective combination therapies for high-grade serous ovarian cancer using patient-derived xenograft models.

Abstract Despite advances in understanding the genetics and molecular biology of high grade serous ovarian cancer, there have not been any significant improvements in the outcome of patients treated with approved therapies to date. The overall aim of our studies is to identify synergistic drug combinations for the treatment of high-grade serous ovarian (HGS-Ov) cancer and the biomarkers that predict sensitivity for future translation in clinical trials. In particular, this study focuses on the vulnerabilities of patient-derived ovarian cancer cells to inhibition of anti-apoptotic proteins and the design of novel combination therapies to overcome drug resistance and improve the response of selected patients to a particular set of targeted therapies. Previous studies from our lab provided evidence that inhibition of BCL-2/XL can significantly enhance the sensitivity of tumor cells to targeted therapies in ovarian and breast cancer cell line models (Muranen T, et al. Cancer Cell 2012). In this study, we evaluated the efficacy of this combination treatment in ascites cells derived from 15 high-grade serous patient-derived ovarian cancer xenografts (PDXs). We found that inhibitors of PI3K/mTOR (GNE-493) and BCL-2/XL (ABT-737) act synergistically ex vivo, with an ~15 fold variability among the 15 patient-derived samples. A large scale in vivo experiment is ongoing to evaluate the efficacy of this combined treatment in six PDX models. To identify biomarkers that predict drug sensitivity we performed proteomic Reverse Phase Protein Array (RPPA) and immunoblot analyses. We found that baseline levels of the pro-apoptotic protein BIM correlate with sensitivity to BCL-2/XL inhibition. This motivated us to examine the basis for low BIM expression. In three of the least sensitive PDX models, we found that low levels of BIM correlated with ERK activation based on increased ERK phosphorylation and that inhibition of MEK by PD-0325901 in these models caused upregulation of BIM, as predicted from previous published reports which demonstrated that phosphorylation of BIM by ERK causes its degradation (Ley R, et al. Biol Chem 2003). We then examined the sensitivity of these PDX models to ABT-737 and PD-0325901, MEK162, or CI-1040 ex vivo and observed strong synergy resulting in reduction of cell viability and increased cell death. These results suggest that low BIM/high phosphoERK is a potential biomarker for sensitivity of ovarian tumors to combined MEK and BCL-2/XL inhibition. We now plan to examine the efficacy of drug combinations that target MEK and BCL-2/XL in these PDX models in vivo. Our studies promise to lead to the identification of new drug combination therapies for HGS-Ov cancer treatment and predictive biomarkers to stratify patients that can benefit from these targeted therapies. Citation Format: Claudia Iavarone, Ioannis Zervantonakis, Hsing-Yu Chen, Sangeetha S. Palakurthi, Joyce Fu Liu, Ursula A. Matulonis, Ronny I. Drapkin, Gordon B. Mills, Joel D. Leverson, Deepak Sampath, Joan S. Brugge. Design of effective combination therapies for high-grade serous ovarian cancer using patient-derived xenograft models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B48.

Abstract A090: Exploring folate receptor α immunotherapy of breast carcinomas: Human monocytic cell-mediated killing triggered by IgG1 and IgE antibodies

Abstract Background: Folate receptor α is a GPI-anchored cell surface protein that mediates the intracellular transport of folates. It is over-expressed in a number of malignancies, including ovarian carcinoma and mesothelioma and has limited normal tissue expression. More recently it has been found to be over-expressed in breast cancer, and particularly a less favourable prognostic form, known as ‘triple-negative’ breast cancer (TNBC). We examined a therapeutic antibody candidate recognising FRα; which we are preparing for Phase 1 clinical trials for its potential to target human immune effector cells against breast cancer cells. As part of this effort, we endeavoured to identify a breast cancer cell line model in which the ability for this antibody to induce Antibody-dependent Cell-mediated breast tumour cell death could be evaluated in vitro. Methods: Twenty-two breast cancer cell lines were evaluated by flow cytometry for surface expression of FRα using the anti-FRα antibody compared with IGROV1, a well characterised ovarian cancer cell line used as a positive control. We confirmed these findings with mRNA levels for cell lines available from the publically accessible Cancer Cell Line Encyclopaedia (CCLE). The two highest expressing breast cancer cell lines, T47D and CAL51 were then studied in a functional flow cytometric assay which can simultaneously measure antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP). The human monocytic U937 cells which express Fc receptors for IgG and IgE antibodies, served as effectors. We compared the capacity of the the anti-FRα antibodies (IgG1 and IgE isotypes) to activate monocytic cells against the FRα-expressing ovarian carcinoma IGROV1 cells, the breast carcinoma T47D and CAL51 cells and the FRα non-expressing breast cancer cell line, HCC1428. Results: There was wide variety in the levels of cell surface expression of FRα; by breast cancer cell lines, expressed as Mean Fluorescent Intensity (>than an isotype control). The highest expressing breast cancer cell lines, T47D and CAL51 had values of 1273 (SEM 13.6) and 1638 (SEM 24.7) respectively in comparison with the non-expressing cell line, HCC1428 with a value of 8 (SEM 0.54). Expression levels in the ovarian cancer cell line, IGROV1 were 16580 (SEM 2534). These values correlated well with mRNA levels from the CCLE database (Spearman's rank, r=0.6011, p=0.0065) which varied from 1x104-1x1011 amongst breast cancer cell lines but were still below those detected with IGROV1 ovarian cancer cells with an mRNA level of 7x1012. We detected no clear evidence of expressing/non-expressing cell lines clustering amongst any known molecular subtype of TNBC. In vitro functional assay assessments of breast carcinoma cell killing in the non-FRα expressing cell line, HCC1428, revealed that neither IgG nor IgE induced breast cancer cell cytotoxicity above that induced by a non-targeting isotype antibody control. In contrast anti-FRα IgG1 and IgE antibodies could induce 20-30% more ADCP and ADCC, respectively, of the FRα+ IGROV1 tumour cells than the isotype control. The breast carcinoma T47D cells, likewise, were killed by ADCP when exposed to anti-FRα IgG, while the anti-FRα IgE antibody induced high levels of ADCC of ~40% above isotype control. Furthermore, treatment of breast carcinoma cells with anti-FRα antibodies induced direct signalling effects that may lead to reduced growth and survival cascades. Conclusions: We have identified two breast cancer cell line models which could be candidates for further in vitro and in vivo assessment of the efficacy and mechanisms of the novel anti-FRα; IgE against breast carcinomas. We have demonstrated similar levels of human immune effector cell-mediated breast cancer cell killing in one of these cell lines, T47D, to a well characterised ovarian cancer cell line, IGROV1, not seen in the FRα; non-expressing cell line, HCC1428. This tumour antigen warrants further characterisation as a potential new immune therapy in TNBC, where immune infiltration is already known to be favourably prognostic. Citation Format: Matthew W. Fittall, Anthony Cheung, Gyula M. Petranyi, Diana Rodriguez-Dominguez, Heather J. Bax, Panagiotis Karagiannis, Kristina M. Ilieva, Andrew Tutt, Sophia N. Karagiannis. Exploring folate receptor α immunotherapy of breast carcinomas: Human monocytic cell-mediated killing triggered by IgG1 and IgE antibodies. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A090.

Abstract B19: LKB1-AMPK axis regulates the switch of Pyruvate Kinase M isoforms to tolerate nutritional stress

Abstract Background: Cancer cells are known to acquire a unique metabolic strategy of aerobic glycolysis (Warburg effect), where they rely more on glycolysis rather than oxidative phosphorylation, regardless of ample amount of oxygen. Aerobic glycolysis is presumed as an inefficient way to generate energy, yet believed to sustain equilibrium between catabolism and anabolism to achieve limitless proliferative competence in cancer cells. Conversely, glucose deprivation is an inevitable characteristic feature of tumors due to their rate of proliferation and poor vasculature. Thus, how cancer cells with an often encountered microenvironment within the tumor of hypoglycemia co-ordinate their growth and proliferates with an abnormal bio-energetics remains ambiguous. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase, known to get activated by sensing bioenergetic stress and remodels the core of cellular metabolism to preserve energy homeostasis. Recent studies have appreciated the critical importance of Liver kinase B1 (LKB1) – AMPK pathway in tumorigenesis, where the axis of LKB1-AMPK has been shown to provide tolerance to nutrient deprived cancer, but how AMPK contributes to cancer cell metabolism is largely unknown, except for the established role in enzymes, like PFK2 (phospho fructokinase 2); and more work is needed to define the role of AMPK in regulation of other essential glycolytic enzymes: Hexokinase (HK), Pyruvate Kinase (M2 PK) and Lactate Dehydrogenase (LDH), associated with aerobic glycolysis. Methodology: Lung cancer (H1299 & A549) and breast cancer (MCF-7 & MDA MB 231) cell lines were used in this study. qPCR and Immunoblotting analysis were performed to measure the gene expression status. Oligomeric status and the distribution of PKM isoforms was assessed using density gradient ultracentrifugation followed by immunoblotting. PKM1 and PKM2 knockdowns were generated by lentiviral transduction. Metabolites levels were assayed using enzyme coupled assays. Cell proliferation was assessed using MTB assay; and apoptosis measured by developing immunoblot for Cleaved PARP and Cleaved Caspase 3. Results: We show how activation of AMPK in cultured cancer cells through relative glucose deprivation (25, 5 & 1 mM) increased the relative activity of PKM, but not of HK and LDH. Further, we demonstrate AMPK facilitates PK activity by enhancing their subunit association, which is the consequential outcome of PKM isoform switch from a catalytically less active M2 isoform to more active M1 isoform. Other than the response in glucose deprivation, the role of AMPK in the PKM isoform switch is confirmed by experimenting with AMPK agonist, inhibitor and constitutively active mutant of AMPK in cell culture. We also show the coordinated axis between LKB1 and AMPK in mediating alternative splicing of PKM isoforms, where transfection of LKB1 cDNA was carried out in cell lines devoid of LKB1. We for the first time report the indirect involvement of AMPK in regulation of alternative splicing of PKM isoforms, independent of c-Myc. Moreover, we show either impairment of AMPK activation through pharmacological agents or knockdown of PKM1 isoform in cancer cells cultured in glucose deprived medium (5mM glucose), diminishes the rate of glucose uptake, lactate production and cellular pyruvate and ATP level, as a result of attenuated cell proliferation and enhanced onset of apoptosis. Together our findings suggest that AMPK –PKM1 axis enhances the aerobic glycolysis and favors cancer cells in culture by conserving the energy homeostasis to endure nutritional stress.” Conclusion: The study highlights the integral role of LKB1-AMPK axis in dynamically regulating the preferential expression of either PKM2 or PKM1 in response to nutritional status in lung cancer and breast cancer cell line models, drawing an inference with regard to real life tumor microenvironment. Citation Format: Gopinath Prakasam, Rameshwar N.K. Bamezai. LKB1-AMPK axis regulates the switch of Pyruvate Kinase M isoforms to tolerate nutritional stress. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B19.

Abstract 680: Identification of GLI1 antagonists for breast cancer therapy

Abstract Introduction: The transcription factor, glioma-associated oncogene homolog 1 (GLI1) is a downstream marker for hedgehog (Hh) pathway activation. Recently, Hh-GLI has emerged as a central pathway target in several human cancers (breast, leukemia, lung etc.) with elevated GLI1 in breast cancer having been linked to poor survival outcomes. A number of GLI targeted inhibitors have been shown to inhibit Hh/GLI signaling with some having efficacy in tumor models. We have previously demonstrated that siRNA downregulation of GLI1 expression in inflammatory breast cancer reduced proliferation and migration[1]. Further, we previously demonstrated the feasibility of employing a high throughput (HT) approach to profile drugs in dose response in cell line panels [2]. In this study, we have taken a comprehensive approach to profile a panel of GLI antagonists (including GANTS, HPIs, ATO and JK184) in a range of breast cancer cell lines with varying GLI1 expression levels to identify those with effects on growth. Experiment procedure: to assess cell proliferation, we employed an automated quantitative high throughput (qHT) approach to profile compounds on three phenotypic subtypes of breast cancer cell line models: triple negative breast cancer (TNBC), inflammatory breast cancer, and Claudin low breast cancer cell lines. High-content imaging of nuclear count utilizing Hoechst staining was also used as an alternative measure of proliferation. Quantitative PCR and Western blotting was used to assess the effects of the inhibitors on the GLI1 expression. Results: A subset of GLI antagonists were identified as decreasing cell growth and viability in all the cell lines. Conclusion: Several agents showed efficacy in in vitro BC cancer models demonstrating that GLI inhibitors may be a valid therapeutic approach for targeting GLI-dependent BC cancers. [1] Z. Thomas, W. Gibson, J. Sexton, K. Aird, S. Ingram, A. Aldrich, H. Lyerly, G. Devi, K. Williams, Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. British Journal of Cancer 104 (2011) 1575-1586. [2] K.P. Williams, J.L. Allensworth, S.M. Ingram, G.R. Smith, A.J. Aldrich, J. Z Sexton, G. R Devi, Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Lett. 337 (2013) 77-89. Funded in part by DOD/CDMRP IDEA W81XWH-13-1-0141 award (KPW); NIH U54CA156735 (KPW); Duke Cancer Institute - Cancer and Environment Initiative P3917733 sub-award (GRD); W81XWH-13-1-041 subcontract (GRD) and National Cancer Institute training grant T32CA009111 (SJS). Citation Format: Helen Oladapo, Jodie M. Fleming, Kezia Addo, Mike Tarpley, Ben Ehe, Shalonda Ingram, Scott Sauer, Gayathri Devi, Kevin P. Williams. Identification of GLI1 antagonists for breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2015-680

Abstract 5038: Role of the short isoform of the progesterone receptor in hormone-regulated invasiveness of breast cancer cells at pre- and post-menopausal levels of estrogen and progesterone

Abstract Introduction: High expression of the progesterone receptor (PR) isoform A, relative to PR isoform B, is clinically associated with progression to invasiveness in estrogen receptor (ER)-positive breast cancer and lower disease free survival. This is apparently at odds with in vitro studies implicating PR-B in mediating progestin-induced invasiveness. As the in vitro studies were conducted in the absence of estrogen (E2) signaling, we considered the possibility that modulation of E2 action may comprise a distinct aspect of the regulation of invasiveness by progestins. Methods: Standard ER+/PR+ and ER+/PR+/HER2+ breast cancer cell line models were studied, as well as recombinant cells expressing a single PR isoform. For the progestin hyper-sensitization studies, PR-A was over-expressed by lentiviral infection. Hormonal influence on invasiveness was studied using the Boyden chamber assay. Gene expression was examined by Illumina DNA microarray analysis and by Taq-Man real time RT-PCR assays. Results: E2 strongly suppressed invasiveness of ER+ breast cancer cells at concentrations below 0.01nM. At low (< 1 nM) concentrations, progesterone, R5020 and medroxyprogesterone acetate completely abrogated the inhibition of invasiveness by E2. It was only in a higher (5nM - 50 nM) concentration range that progestins induced invasiveness in the absence of E2. The ability of progestins to rescue invasiveness from E2 regulation was exclusively mediated by PR-A whereas PR-B mediated the E2-independent component of progestin-induced invasiveness. Overexpression of PR-A in PR-A+/PR-B+ cells lowered the progestin concentration needed to completely rescue invasiveness (to < 0.2 nM). Progesterone opposed E2 regulation of a distinctive set of genes, exclusively through PR-A. Conclusion: At relatively low concentrations, progesterone influences breast cancer cell invasiveness by rescuing it from E2 regulation via PR-A, whereas at higher concentrations the hormone also induces invasiveness independent of E2 signaling, through PR-B. The findings establish a direct functional link between PR-A and progression of luminal breast cancer in the context of the entire range of pre- and post-menopausal plasma and breast tissue estrogen and progesterone levels. Citation Format: Thomas B. McFall, Mugdha Patki, Rayna Rosati, Manohar Ratnam. Role of the short isoform of the progesterone receptor in hormone-regulated invasiveness of breast cancer cells at pre- and post-menopausal levels of estrogen and progesterone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5038. doi:10.1158/1538-7445.AM2015-5038

Abstract 737: Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance

Abstract Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a subset of pre-treatment specimens from TBCRC006. Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (>100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed. Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ∼30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ∼30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC50: Afa 0.02μM vs. L 3 μM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation. Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura Heiser, Nicholas Wang, Kyle Covington, Edward Chen, Alexander Renwick, Tamika Mitchell, Marty Shea, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne Fuqua, Gary Chamness, Chad Shaw, Marilyn Li, David Wheeler, Susan Hilsenbeck, Mothaffar Fahed Rimawi, Joe Gray, C.Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 737. doi:10.1158/1538-7445.AM2015-737

A Role for Histone H2B Variants in Endocrine-Resistant Breast Cancer.

Acquired resistance to aromatase inhibitors (AIs) remains a major clinical problem in the treatment of estrogen receptor-positive (ER+) breast cancer. We and others have previously reported widespread changes in DNA methylation using breast cancer cell line models of endocrine resistance. Here, we show that the histone variant HIST1H2BE is hypomethylated in estrogen deprivation-resistant C4-12 and long-term estrogen-deprived (LTED) cells compared with parental MCF-7 cells. As expected, this hypomethylation associates with increased expression of HIST1H2BE in C4-12 and LTED cells. Both overexpression and downregulation of HIST1H2BE caused decreased proliferation in breast cancer cell lines suggesting the need for tightly controlled expression of this histone variant. Gene expression analysis showed varied expression of HIST1H2BE in a large panel of breast cancer cell lines, without restriction to specific molecular subtypes. Analysis of HIST1H2BE messenger RNA (mRNA) expression in ER+ AI-treated breast tumors showed significantly higher expression in resistant (n = 19) compared with sensitive (n = 37) tumors (p = 0.01). Using nanostring analysis, we measured expression of all 61 histone variants in endocrine-resistant and endocrine-sensitive tumors. We found significant overexpression of 22 variant histone genes in tumors resistant to AI therapy. In silico The Cancer Genome Atlas (TCGA) analysis showed frequent amplification of the HIST1 locus. In summary, our studies show, for the first time, that overexpression of histone variants might be important in endocrine response in ER+ breast cancer, and that overexpression is at least in part mediated via epigenetic mechanisms and amplifications. Future studies addressing endocrine response should include a potential role of these currently understudied histone variants.

MicroRNA profiling of the pubertal mouse mammary gland identifies miR-184 as a candidate breast tumour suppressor gene.

IntroductionThe study of mammalian development has offered many insights into the molecular aetiology of cancer. We previously used analysis of mammary morphogenesis to discover a critical role for GATA-3 in mammary developmental and carcinogenesis. In recent years an important role for microRNAs (miRNAs) in a myriad of cellular processes in development and in oncogenesis has emerged.MethodsmicroRNA profiling was conducted on stromal and epithelial cellular subsets microdissected from the pubertal mouse mammary gland. miR-184 was reactivated by transient or stable overexpression in breast cancer cell lines and examined using a series of in vitro (proliferation, tumour-sphere and protein synthesis) assays. Orthotopic xenografts of breast cancer cells were used to assess the effect of miR-184 on tumourigenesis as well as distant metastasis. Interactions between miR-184 and its putative targets were assessed by quantitative PCR, microarray, bioinformatics and 3′ untranslated region Luciferase reporter assay. The methylation status of primary patient samples was determined by MBD-Cap sequencing. Lastly, the clinical prognostic significance of miR-184 putative targets was assessed using publicly available datasets.ResultsA large number of microRNA were restricted in their expression to specific tissue subsets. MicroRNA-184 (miR-184) was exclusively expressed in epithelial cells and markedly upregulated during differentiation of the proliferative, invasive cells of the pubertal terminal end bud (TEB) into ductal epithelial cells in vivo. miR-184 expression was silenced in mouse tumour models compared to non-transformed epithelium and in a majority of breast cancer cell line models. Ectopic reactivation of miR-184 inhibited the proliferation and self-renewal of triple negative breast cancer (TNBC) cell lines in vitro and delayed primary tumour formation and reduced metastatic burden in vivo. Gene expression studies uncovered multi-factorial regulation of genes in the AKT/mTORC1 pathway by miR-184. In clinical breast cancer tissues, expression of miR-184 is lost in primary TNBCs while the miR-184 promoter is methylated in a subset of lymph node metastases from TNBC patients.ConclusionsThese studies elucidate a new layer of regulation in the PI3K/AKT/mTOR pathway with relevance to mammary development and tumour progression and identify miR-184 as a putative breast tumour suppressor.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0593-0) contains supplementary material, which is available to authorized users.

ERRγ target genes are poor prognostic factors in Tamoxifen-treated breast cancer.

BackgroundOne-third of estrogen (ER+) and/or progesterone receptor-positive (PGR+) breast tumors treated with Tamoxifen (TAM) do not respond to initial treatment, and the remaining 70% are at risk to relapse in the future. Estrogen-related receptor gamma (ESRRG, ERRγ) is an orphan nuclear receptor with broad, structural similarities to classical ER that is widely implicated in the transcriptional regulation of energy homeostasis. We have previously demonstrated that ERRγ induces resistance to TAM in ER+ breast cancer models, and that the receptor’s transcriptional activity is modified by activation of the ERK/MAPK pathway. We hypothesize that hyper-activation or over-expression of ERRγ induces a pro-survival transcriptional program that impairs the ability of TAM to inhibit the growth of ER+ breast cancer. The goal of the present study is to determine whether ERRγ target genes are associated with reduced distant metastasis-free survival (DMFS) in ER+ breast cancer treated with TAM.MethodsRaw gene expression data was obtained from 3 publicly available breast cancer clinical studies of women with ER+ breast cancer who received TAM as their sole endocrine therapy. ERRγ target genes were selected from 2 studies that published validated chromatin immunoprecipitation (ChIP) analyses of ERRγ promoter occupancy. Kaplan-Meier estimation was used to determine the association of ERRγ target genes with DMFS, and selected genes were validated in ER+, MCF7 breast cancer cells that express exogenous ERRγ.ResultsThirty-seven validated receptor target genes were statistically significantly altered in women who experienced a DM within 5 years, and could classify several independent studies into poor vs. good DMFS. Two genes (EEF1A2 and PPIF) could similarly separate ER+, TAM-treated breast tumors by DMFS, and their protein levels were measured in an ER+ breast cancer cell line model with exogenous ERRγ. Finally, expression of ERRγ and these two target genes are elevated in models of ER+ breast cancer with hyperactivation of ERK/MAPK.ConclusionsERRγ signaling is associated with poor DMFS in ER+, TAM-treated breast cancer, and ESRRG, EEF1A2, and PPIF comprise a 3-gene signaling node that may contribute to TAM resistance in the context of an active ERK/MAPK pathway.

Abstract P1-06-04: Super-enhancer analysis defines breast cancer subtype and identifies tumor dependencies

Abstract Epigenomic modifications define gene regulatory features that control transcription and disease cell state. Recent studies of these regulatory features have identified large clusters of enhancers, termed super-enhancers, which define key cell identity and disease genes. Using ChIP-seq and RNA-seq analysis, we have discovered Super-enhancers in breast cancer cell line models and in primary tissue and have characterized their roles in establishing tumor cell state. We find that Super-enhancers recapitulate clinical subgroups in both breast cancer cell line models and in invasive ductal carcinoma. Super-enhancer-associated genes encode known and novel therapeutic targets including kinases, phosphatases, chromatin regulators and transmembrane proteins. Such genes include key drivers such as ERRB2 in HER2+ patient samples, ESR1 in estrogen receptor positive samples, and CCND1 in samples of luminal subtype. We describe the biological and disease relevance of Super-enhancer-associated genes in the context of tumor cell state and drug target discovery. Citation Format: Matthew G Guenther, David A Orlando, Matthew L Eaton, Cindy A Collins, Mei Wei Chen, Sneha Solanki, Jakob Loven, Christian C Fritz, Eric R Olson. Super-enhancer analysis defines breast cancer subtype and identifies tumor dependencies [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-04.

Abstract P5-05-03: Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib

Abstract Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-03.