Breast Cancer Cell Line Models Research Articles

GPCRs profiling and identification of GPR110 as a potential new target in HER2+ breast cancer.

G protein-coupled receptors (GPCRs) represent the largest family of druggable targets in human genome. Although several GPCRs can cross-talk with the human epidermal growth factor receptors (HERs), the expression and function of most GPCRs remain unknown in HER2+ breast cancer (BC). In this study, we aimed to evaluate gene expression of GPCRs in tumorigenic or anti-HER2 drug-resistant cells and to understand the potential role of candidate GPCRs in HER2+ BC. Gene expression of 352 GPCRs was profiled in Aldeflur+ tumorigenic versus Aldeflur- population and anti-HER2 therapy-resistant derivatives versus parental cells of HER2+ BT474 cells. The GPCR candidates were confirmed in 7 additional HER2+ BC cell line models and publicly available patient dataset. Anchorage-dependent and anchorage-independent cell growth, mammosphere formation, and migration/invasion were evaluated upon GPR110 knockdown by siRNA in BT474 and SKBR3 parental and lapatinib+ trastuzumab-resistant (LTR) cells. Adhesion and class A GPCRs were overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population of BT474 cells, respectively. GPR110 was the only GPCR overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population in BT474, SKBR3, HCC1569, MDA-MB-361, AU565, and/or HCC202 cells and in HER2+ BC subtype in patient tumors. Using BT474 and SKBR3 parental and LTR cells, we found that GPR110 knockdown significantly reduced anchorage-dependent/independent cell growth as well as migration/invasion of parental and LTR cells and mammosphere formation in LTR derivatives and not in parental cells. Our data suggest a potential role of GPR110 in tumorigenicity and in tumor cell dissemination in HER2+ BC.

Cluster Analysis of Endogenous HER2 and HER3 Receptors in SKBR3 Cells.

The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the in-situ organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters. Clustering of receptors is an important regulatory mechanism to prime cells to respond to stimuli so, to understand these processes, it is necessary to measure parameters such as numbers of clusters, cluster radii and the number of localizations per cluster for different perturbations. Previously, Fluorescence Localization Imaging with Photobleaching (FLImP), another nanoscale, single-molecule technique, characterized the oligomerization state of HER1 [or Epidermal Growth Factor Receptors (EGFR)] in cell membranes. To achieve an unprecedented resolution (< 5 nm) for inter-molecular separations in EGFR oligomers using FLImP, very few receptors are tagged, and so this method is unsuitable for measurements of whole receptor populations in cancer cells where receptors are frequently upregulated. Here, in order to detect all receptors involved in cluster formation, we saturate endogenous HER2 and HER3 membrane receptors with ligands at a 1:1 dye to protein ratio, in the presence or absence of therapeutic drugs (lapatinib or bosutinib). This is performed in the commonly used breast cancer cell line model SKBR3 cells, where there are ~1.6 million HER2 receptors/cell and 10,000-40,000 HER3 receptors/cell. The basal state of these receptors is studied using HER2- or HER3-specific Affibodies, and likewise, the active state is probed using the natural HER3 ligand, Neuregulin-beta1 (NRGβ1). Stochastic Optical Reconstruction Microscopy (STORM), one form of SMLM, was used here to image cells, which were chemically fixed to minimize image blurring and provide data (x and y coordinates and standard deviation of the measured localizations) for cluster analysis. Further analysis can also determine proportions of receptor colocalizations. Our findings show that lapatinib-bound HER2, complexed with HER3 via a non-canonical kinase dimer structure, induces higher order oligomers. We hypothesized that nucleation of receptors creates signaling platforms that explain the counterintuitive, increase in cell proliferation upon ligand binding, in the presence of the HER2-inhibitor lapatinib.

Interferon-Stimulated Genes Are Transcriptionally Repressed by PR in Breast Cancer.

The progesterone receptor (PR) regulates transcriptional programs that drive proliferation, survival, and stem cell phenotypes. Although the role of native progesterone in the development of breast cancer remains controversial, PR clearly alters the transcriptome in breast tumors. This study identifies a class of genes, Interferon (IFN)-stimulated genes (ISGs), potently downregulated by ligand-activated PR which have not been previously shown to be regulated by PR. Progestin-dependent transcriptional repression of ISGs was observed in breast cancer cell line models and human breast tumors. Ligand-independent regulation of ISGs was also observed, as basal transcript levels were markedly higher in cells with PR knockdown. PR repressed ISG transcription in response to IFN treatment, the canonical mechanism through which these genes are activated. Liganded PR is robustly recruited to enhancer regions of ISGs, and ISG transcriptional repression is dependent upon PR's ability to bind DNA. In response to PR activation, key regulatory transcription factors that are required for IFN-activated ISG transcription, STAT2 and IRF9, exhibit impaired recruitment to ISG promoter regions, correlating with PR/ligand-dependent ISG transcriptional repression. IFN activation is a critical early step in nascent tumor recognition and destruction through immunosurveillance. As the large majority of breast tumors are PR positive at the time of diagnosis, PR-dependent downregulation of IFN signaling may be a mechanism through which early PR-positive breast tumors evade the immune system and develop into clinically relevant tumors.Implications: This study highlights a novel transcriptional mechanism through which PR drives breast cancer development and potentially evades the immune system. Mol Cancer Res; 15(10); 1331-40. ©2017 AACR.

14P - Alterations to trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (T-ADCC) in a lapatinib-resistant HER2+ breast cancer cell line model

14P - Alterations to trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (T-ADCC) in a lapatinib-resistant HER2+ breast cancer cell line model

Abstract 5499: The reprogramming of the steroid receptor binding landscape at active enhancers is associated with a fast DNA residence time through a mechanism termed dynamic assisted loading in breast cancer cells

Abstract The transcription factor (TF), FoxA1 has been described as a pioneer factor, implicated in steroid receptor (SR) binding patterns. Classically it has been proposed that the pioneer factor model is a slow and static binding event. However, it has recently been described that the estrogen and glucocorticoid receptors (ER and GR), can regulate the binding patterns of each other in a mouse mammary cell line defined as dynamic assisted loading. Further, this phenomenon has been extending demonstrating that both ER and GR can recruit FoxA1 to specific binding sites associated with an increase in chromatin accessibility and fast DNA residence times. This was also associated with the absence of a DNaseI footprint for the initiating and secondary factor supporting the assisted loading model. To further investigate the molecular mechanism behind the dynamic assisted loading model we have characterized the binding and transcriptional landscape of ER and GR in a number of breast cancer cell line models. Genome wide analysis of ChIP and DNaseI hypersensitivity assays upon single and dual activation show that both SRs can recruit each other to a specific subset of binding sites associated with an increase in chromatin accessibility. Therefore, depending on the chromatin landscape, one factor functions as the initiating factor and the other the secondary factor. Further, RNA polymerase II (RNAP II), p300, and H3K27ac binding patterns are associated with the newly programed ER or GR binding sites. Together this suggests chromatin reorganization upon dual activation of either ER or GR (the initiating factors) and recruitment of the secondary factor (ER or GR) through an assisted loading mechanism. Further, we have demonstrated an association of the transcriptional machinery at these newly acquired binding sites at active enhancers. These results propose that many TFs in a given cell have the potential to affect the binding landscape of other TFs, depending on the chromatin context. In addition, this study has shifted our classical understanding of pioneer factors in breast cancer, demonstrating that not only can SRs alter the response of FoxA1 but also each other though highly dynamic and fast DNA interactions associated with active enhancers and transcription. Citation Format: Erin Swinstead, Tina Miranda, Ville Paakinaho, Songjoon Beak, Gordon Hager. The reprogramming of the steroid receptor binding landscape at active enhancers is associated with a fast DNA residence time through a mechanism termed dynamic assisted loading in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5499. doi:10.1158/1538-7445.AM2017-5499

Abstract 5769: Modeling the tumor invasion front using 3D fluidic tumoroid culture of cancer cells

Abstract To realize the promise of precision medicine, it is important to integrate phenotypic assessment of cell populations to genomic data. The analysis of invading leader cells at the tumor invasion front is of interest as they may be guided by a targetable molecular phenotype. However, there is a lack of suitable platforms on which to analyze the tumor invasion front. In this study, we have designed and constructed a fluidic device for long-term (several days to weeks) 3-dimensional tumoroid culture of diverse cancer cells. Using this device, we can recapitulate the tumor invasion front and at the same time quantify the invasive potential of different breast cancer cell line models. Analyses of the tumor invasion front indicated a region of higher proliferation and suggest that the leader cells possess a different molecular phenotype from the tumoroid mass. Interestingly, significant heterogeneity among invading cells was still observed, suggesting that there could be: 1) the presence of multiple subpopulations of invasive cells, each with a different clonal genetic signature; and/or 2) reversible phenotypic switching occurring among invading cells due to phenotypic plasticity. These results obtained using this innovative device highlight and present a promising solution to the challenges developing adequate therapeutics accounting for tumor phenotypic heterogeneity. There is potential for the device for use in personalized medicine at diagnosis, allowing for both the quantification of disease progression risk as well as the molecular characterization of the invasive subpopulations from patient samples and their response to tailored therapies. Citation Format: Koh Meng Aw Yong, Christopher Oliver, Megan Altemus, Zhi Fen Wu, Sofia Merajver. Modeling the tumor invasion front using 3D fluidic tumoroid culture of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5769. doi:10.1158/1538-7445.AM2017-5769

Inhibition of Inositol 1, 4, 5-Trisphosphate Receptor Induce Breast Cancer Cell Death Through Deregulated Autophagy and Cellular Bioenergetics.

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) regulate autophagy in normal cells and are associated with metastasis in cancer cells. In breast cancer, however, the regulation and role of IP3 Rs is not clear. To study this, we used MCF-7 breast cancer cell line and mouse model of breast cancer. Inhibiting IP3 R sub types resulted in compromised bioenergetics both in terms of glucose and mitochondrial metabolism. The siRNA mediated silencing of IP3 R or its blocking by its inhibitors Xestospongin C and 2-Amino-ethoxy diphenyl borate increased cell death and LC3II expression in MCF-7 cells as well as attenuated cellular bioenergetics. The level of Autophagy related gene, Atg5 was found to be up regulated after pharmacological as well as siRNA blocking of IP3 R. The specificity of its role in autophagy was confirmed through specific shRNA knockdown of the Atg5 along with IP3 R inhibitor. Inhibiting as well as silencing of IP3 R receptor also resulted in increase in ROS production which was abolished after pretreatment with N-acetyl cysteine. Its role in autophagy was confirmed through decrease in the levels of LC3 II after pretreatment with IP3 R inhibitor and N acetyl cysteine.Moreover, inhibiting as well as silencing IP3 R-induced cell death in MCF-7 cells was attenuated by autophagic inhibitors (Bafilomycin A1 or 3-Methyladeneine). In mice, blocking of IP3 Rs by 2-Amino-ethoxy diphenyl borate arrested tumor growth. Overall our findings indicate that IP3 R blocking resulted in autophagic cell death in breast cancer cells and provides a role of IP3 Rs in determining the breast cancer cell fate. J. Cell. Biochem. 118: 2333-2346, 2017. © 2017 Wiley Periodicals, Inc.

PACE4 is an important driver of ZR-75-1 estrogen receptor-positive breast cancer proliferation and tumor progression

Breast cancer is the most frequent and deadly malignancy in women worldwide. Despite national screening programs combined with new treatments relapse rate remain high and new therapies are needed. From previous work, we identified PACE4, a member of the proprotein convertase (PCs) family of endoproteases, as a novel therapeutic target in prostate cancer. In the present study we asked the question if PACE4 could also be a potential target in breast cancer. In clinical samples of breast adenocarcinoma, we observed a specific overexpression of PACE4 in the estrogen-receptor (ER) positive subtype. We therefore looked for a breast cancer cell line model which would be representative and thus focused on the ZR-75-1 since it both expresses PACE4 and is estrogen-receptor positive. We compared stable knockdowns of furin, PACE4 and PC7 in the estrogen-receptor-positive cell line ZR-75-1 to evaluate their respective contribution to cell growth and tumor progression. PACE4 was the only PC displaying an impact on cell growth. A PACE4 peptide-based inhibitor (C23) was tested and shown to decrease proliferation of ZR-75-1 cells in cell based assays. C23 also had potent effects of tumor progression in vivo on xenografts of the ZR-75-1 cell line in athymic nude mice. Thus, PACE4-silencing and systemic administration of a PACE4 inhibitor resulted in hindered tumor progression with reduction in proliferative indices and increased cell quiescence assessed with biomarkers. Our results suggest that PACE4 is a promising target for estrogen-receptor-positive breast cancer.

Abstract P4-07-06: MicroRNAs associated with acquired taxane resistance in a breast cancer cell line model

Abstract Background: Acquired chemoresistance remains the major cause of therapeutic failure in the treatment of breast cancer. Improved knowledge of the transition from drug sensitive to resistant breast cancer will pave the way for novel preventative and therapeutic strategies. MicroRNAs (miRNA) are endogenous, small non-coding RNAs that regulate gene expression by targeting the 3'UTR region of messenger RNAs. There is a growing body of evidence to suggest miRNAs may be involved in the development of chemoresistance and may play a role in the regulation of drug resistance pathways. Methods: An in vitro model of paclitaxel resistance was developed through the generation of resistant MDA-MB-231 cell lines by serial culture in escalating doses of taxane until resistance was achieved. The chemoresistant model was used to compare differential miRNA expression with the sensitive, parental line using the Nanostring® platform, analysing 800 human miRNAs. Confirmation of differential expression was performed by QRT-PCR. Results: This analysis resulted in 30 significantly altered miRNA (1.5 fold, p value &amp;lt; 0.05) at 25nM paclitaxel and 48 significantly altered miRNA at 50nM paclitaxel. The top up-regulated miRNA cluster in MDA-MB-231 25PACR is hsa-miR-548l (fold change: 2.89, p value: 0.016) and top down-regulated was hsa-miR-449a (fold change: -4.1. p value: 0.001). In MDA-MB-231 50PACR the top up-regulated miRNA cluster is hsa-miR-193a-5p (fold change: 3.746, p value: 0.008) and the top down-regulated miRNA cluster is hsa-miR-135a (fold change: -4.085, p value: 0.001). To explore the molecular mechanisms of the differentially expressed miRNAs in paclitaxel resistance, targets were predicted by in silico analysis. Pathways and networks designated by miRNA targets included the cell cycle, PI3K/Akt pathways and focal adhesion. Conclusion: In this study we identified candidate resistance-associated miRNAs which were differentially expressed between in vitro derived paclitaxel resistant MDA-MB-231 and the sensitive parental line. Further validation to ascertain their role in the transition to a chemoresistant phenotype is currently ongoing. Citation Format: Taylor KJ, Chong T, D'Costa A, Yao C, Gourley C, Cameron DA, Bartlett JMS, Spears M. MicroRNAs associated with acquired taxane resistance in a breast cancer cell line model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-06.

A New Role for ERα: Silencing via DNA Methylation of Basal, Stem Cell, and EMT Genes.

ERα directs DNA methylation-mediated silencing of specific genes that have biomarker potential in breast cancer subtypes. Mol Cancer Res; 15(2); 152-64. ©2016 AACR.

Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration

PurposeMolecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays.MethodsBM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression.ResultsFiltering an average of 14×106 nucleated BM cells yielded approximately 17–21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84–92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM.ConclusionsWe have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.

BRAF, KRAS and PIK3CA Mutation and Sensitivity to Trastuzumab in Breast Cancer Cell Line Model

Studies show that approximately 20% of all breast cancer patients have a breast tumor that tests positive for Human Epidermal Growth Factor Receptor 2, otherwise known as the HER2 gene. As such, treatments for breast cancer usually include drugs that target HER2. The drug Trastuzumab is a recombinant antibody that has been approved by the FDA for the treatment of these HER2 positive breast cancers. However, researchers have found that mutations in associated genes, PIK3CA and KRAS, can cause the tumor to become resistant to Trastuzumab. The purpose of this article is to evaluate the sensitivity of the cancer cell lines to the drug Trastuzumab and investigate how this sensitivity is compromised by the PIK3CA, KRAS and BRAF gene mutations. Trastuzumab responsiveness was evaluated in breast cancer cell lines by treating the lines with an optimal concentration of the drug followed by a proliferation assay of the cell lines in the presence of monoclonal antibodies. We determined the optimum concentration of Trastuzumab to be 7 μg/well. The BRAF and KRAS mutated cell line, MDA-MB-231, showed the least sensitivity after being treated with trastuzumab when compared to the sensitivity of the PIK3CA mutated cell lines, MCF-7 and MDA-MB-361, and the KRAS/ BRAF/ PIK3CA cell line, MDA-MB-453. Clinical observations show that mutations in BRAF and KRAS genes in breast cancer cells do lower the responsiveness of Trastuzumab drug treatments.

FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer

Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)-chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

Sperm-associated antigen 9 (SPAG9) promotes the survival and tumor growth of triple-negative breast cancer cells.

Recently, we demonstrated the association of sperm-associated antigen 9 (SPAG9) expression with breast cancer. Among breast cancer, 15% of the cancers are diagnosed as triple-negative breast cancers (TNBC) based on hormone receptor status and represent an important clinical challenge because of lack of effective available targeted therapy. Therefore, in the present investigation, plasmid-based small hairpin (small hairpin RNA (shRNA)) approach was used to ablate SPAG9 in aggressive breast cancer cell line model (MDA-MB-231) in order to understand the role of SPAG9 at molecular level in apoptosis, cell cycle, and epithelial-to-mesenchymal transition (EMT) signaling. Our data in MDA-MB-231 cells showed that ablation of SPAG9 resulted in membrane blebbing, increased mitochondrial membrane potential, DNA fragmentation, phosphatidyl serine surface expression, and caspase activation. SPAG9 depletion also resulted in cell cycle arrest in G0-G1 phase and induced cellular senescence. In addition, in in vitro and in vivo xenograft studies, ablation of SPAG9 resulted in upregulation of p21 along with pro-apoptotic molecules such as BAK, BAX, BIM, BID, NOXA, AIF, Cyto-C, PARP1, APAF1, Caspase 3, and Caspase 9 and epithelial marker, E-cadherin. Also, SPAG9-depleted cells showed downregulation of cyclin B1, cyclin D1, cyclin E, CDK1, CDK4, CDK6, BCL2, Bcl-xL, XIAP, cIAP2, MCL1, GRP78, SLUG, SNAIL, TWIST, vimentin, N-cadherin, MMP2, MMP3, MMP9, SMA, and β-catenin. Collectively, our data suggests that SPAG9 promotes tumor growth by inhibiting apoptosis, altering cell cycle, and enhancing EMT signaling in in vitro cells and in vivo mouse model. Hence, SPAG9 may be a potential novel target for therapeutic use in TNBC treatment.

Abstract 319: Role of GPR110 on tumorigenesis and metastasis in HER2+ breast cancer in the context of anti-HER2 drug resistance

Abstract Drug resistance is a major limitation of treatment regimens targeting the human epidermal growth factor receptor-2 (HER2) in breast cancers that overexpress this protein. Therefore, identification of novel drug targets to overcome anti-HER2 drug resistance is an unmet clinical need. Our previous studies have suggested that GPR110 may be a potential target for anti-HER2 drug resistance and tumorigenicity. Furthermore, GPR110 is an orphan receptor in the adhesion G-protein coupled receptor family, members of which have shown a role in cancer metastasis. In this study, our objectives were (1) to assess the gene expression of GPR110 using the Taqman RT-PCR assay in anti-HER2 drug resistant and in cancer stem-like cell population of various HER2+ breast cancer cell line models and (2) to investigate the effects of GPR110 gene knockdown on tumorigenesis and metastasis using various cell-based assays in HER2+ breast cancer cell lines and their anti-HER2-resistant derivatives. Cells transfected with non-targeting and 3 independent GPR110-targeting siRNAs were used to assess anchorage-independent cell growth, mammosphere formation, and cell invasion and migration. We found that GPR110 gene expression was significantly higher (&amp;gt;/ = 2-fold) in lapatinib-resistant (LR), trastuzumab-resistant (TR), and lapatinib + trastuzumab-resistant (LTR) derivatives of BT474, SKBR3 and UACC812 cell lines compared to the parental cells. GPR110 gene expression was also significantly higher (&amp;gt;/ = 1.9-fold) in ALDH+ versus ALDH- cell populations of BT474, SKBR3, MDA-MB-361 and HCC1569 cell lines. In addition, GPR110 gene knockdown led to a 55-85% (p&amp;lt;0.05) decrease in anchorage-independent cell growth in BT474 and SKBR3 LTR derivatives but not in parental cells. A significant reduction in mammosphere count upon GPR110 knockdown was also seen in LTR derivatives (50-70%, p&amp;lt;0.05) and not in parental BT474 cells. Interestingly, GPR110 knockdown in SKBR3 parental cells resulted in a significant reduction in cell invasion (40-50%, p&amp;lt;0.05) and migration (40-50%, p&amp;lt;0.05) suggesting a potential role of GPR110 in tumor cell dissemination. Overall, these results demonstrate that GPR110 is overexpressed in multiple anti-HER resistant derivatives as well as breast cancer stem-like cell population of various HER2+ breast cancer cell line models. Our studies also suggest a role of GPR110 in tumorigenesis specifically in the context of anti-HER2 drug resistance and in metastatic processes. Future in vivo studies will assess GPR110 as a novel drug target to overcome anti-HER2 drug resistance and combat metastasis in HER2+ breast cancer. Citation Format: Debashish Sahay, Raksha Bhat, Motthafar Rimawi, C. Kent Osborne, Rachel Schiff, Meghana V. Trivedi. Role of GPR110 on tumorigenesis and metastasis in HER2+ breast cancer in the context of anti-HER2 drug resistance. [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 319.

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, &amp;lt; 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, &amp;gt;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 &amp;lt;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.