Luminal Breast Cancer Cells Research Articles

Aberrant fucosylation enables breast cancer clusterin to interact with dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN)

ABSTRACTClusterin is a glycoprotein able to mediate different physiological functions such as control of complement activation, promotion of unfolded protein clearance and modulation of cell survival. Clusterin is overexpressed in many types of cancers and a large body of evidence suggests that it promotes carcinogenesis and tumor progression. We have previously described a novel clusterin glycoform present in human semen, but not in serum, highly enriched in terminal fucose motifs. Here we show that human luminal breast cancer (LBC) clusterin also bears terminal fucosylated glycans, conferring clusterin the ability to interact with DC-SIGN, a C-type lectin receptor expressed by myeloid cells. This clusterin glycosylation pattern was absent or diminished in non-involved juxtatumoral tissue, suggesting that fucosylated clusterin might represent a cancer associated glycoform. We also found that DC-SIGN is expressed by luminal breast cancer intratumoral macrophages. Moreover, experiments performed in vitro using semen fucosylated clusterin and monocyte derived macrophages showed that the interaction of semen clusterin with DC-SIGN promoted a proangiogenic profile, characterized by a high production of VEGF, IL-8 and TNF-α. Our results reveal an unexpected complexity on the structure and function of secretory clusterin produced by tumors and suggest that fucosylated clusterin produced by luminal breast cancer cells might play a role in tumor progression by promoting the release of pro-angiogenic factors by intratumoral macrophages.

Loss of PICH promotes chromosome instability and cell death in triple-negative breast cancer

Triple-negative breast cancer (TNBC), defined by the lack of expression of estrogen, progesterone, and ERBB2 receptors, has the worst prognosis of all breast cancers. It is difficult to treat owing to a lack of effective molecular targets. Here, we report that the growth of TNBC cells is exceptionally dependent on PICH, a DNA-dependent ATPase. Clinical samples analysis showed that PICH is highly expressed in TNBC compared to other breast cancer subtypes. Importantly, its high expression correlates with higher risk of distal metastasis and worse clinical outcomes. Further analysis revealed that PICH depletion selectively impairs the proliferation of TNBC cells, but not that of luminal breast cancer cells, in vitro and in vivo. In addition, knockdown of PICH in TNBC cells induces the formation of chromatin bridges and lagging chromosomes in anaphase, frequently resulting in micronucleation or binucleation, finally leading to mitotic catastrophe and apoptosis. Collectively, our findings show the dependency of TNBC cells on PICH for faithful chromosome segregation and the clinical potential of PICH inhibition to improve treatment of patients with high-risk TNBC.

Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer

BackgroundEukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy.MethodsHere we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1.ResultsUsing a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes.ConclusionsThese results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

ARID1a as a marker of prognosis and increased sensitivity to CDK4/6, mTOR 1/2 and Src homology region 2 phosphatase (SHP 1/2) inhibitors in breast cancer (BC).

1082 Background: ARID1a (AT Rich Interactive Domain 1A) is part of the SWI/SNF complex, which regulates gene transcription, and is believed to be a tumor suppressor gene. Low ARID1a expression has been associated with poor prognosis in BC. The aim of this study was to explore the clinical significance of ARID1a mutation and expression loss, and its potential as a therapeutic target in BC. Methods: We analyzed publicly available genomic databases to study the clinical implication of ARID1a mutations and gene expression in BC. Results: ARID1a was mutated in ~5-7 % of BCs within TCGA/METABRIC/MSK (5511 samples), but did not show differences in frequency between histology, grade, or estrogen receptor (ER)/HER2 receptor status. MSK metastatic tissue samples had higher incidence of ARID1a mutation compared to primary tumor samples (7.6% vs 4.4%, χ2 P = 0.0073). Analysis of ARID1a in KMPLOT showed that lower gene expression was associated with worse relapse-free survival and overall survival across all BCs, but the difference was primarily in molecularly classified luminal A tumors. Mutations in ARID1a did not show an association with outcomes in TCGA/METABRIC/MSK datasets. Pathway analysis of ARID1a showed it is involved in regulating ER ligand driven signaling and interacts with targets regulated by CDK4 and mTOR activity. CancerRxgene drug sensitivity analyses on BC cell lines revealed that ARID1a mutated BC cell lines were significantly more sensitive to palbociclib, SHP1/2, and mTOR1/2 inhibitors compared to ARID1a wildtype cell lines. Conclusions: Reduced activity of ARID1a in luminal BC cells may negatively affect prognosis by altering ER signaling leading to activation of druggable resistance mechanisms, particularly in metastatic tissue. Loss of function ARID1a mutations may sensitize cancer cells to CDK4/6, mTOR1/2, and SHP1/2 inhibitors in vitro. Further research in ARID1a mutated ER+ BCs using combinations of these inhibitors is warranted.

RERG involvement in the RAS pathway and ER-dependent transcription in breast cancer.

e14638 Background: Breast cancer is the highest incidence malignancies in women worldwide. Luminal breast cancers are typically estrogen receptor–positive with better prognosis. However, the rapid disease progression and the high relapse rate of this subtype of breast cancer have become a puzzle in breast cancer treatment. It gave us the motivation to figure out the anomalous molecular mechanism which devoted in. It has been well-documented that the RAS pathway is overloadly activated in more than half of human breast tumors. RAS genes encode a superfamily of small GTPases that contribute to cell growth signals. Methods: Gene expression level were measured by qPCR and Western Blot.Biological effects of each condition were evaluated in cell viability and migration. Results: In this study, we investigated one member of the RAS family, RERG, which was related to the ER pathway and contributed to inhibit Ras activated pathway. Our results showed that knockdown of RERG concomitantly promoted two major oncogenic pathways, Ras and Stat3 signaling pathways, in luminal type breast cancer cell lines. Moreover, ectopic RERG expression significantly inhibited Ras expression. It implicated that RERG mediated in RAS-driven biological effects. Our findings indicated that knockdown of RERG enhanced mobility of the breast cancer cells and made cells more intractable under SERM treatment. Conclusions: We elucidated the tumor-suppressor role of RERG in breast cancer cells though inhibition of the Ras and Stat3 signaling pathways. Therefore, this study might shed light on the important mechanistic insight into the tumorigenesis of ER-positive luminal type cancer and provided the prognostic and therapeutic improved roles of RERG.

Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer

BackgroundCurrent prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). Building upon current knowledge, we hypothesized that epoxyeicosatrienoic acids (EETs) and related CYP450 epoxygenases may have differential roles in breast cancer signaling, and better understanding of which may uncover potential directions for molecular stratification and personalized therapy for TNBC patients.MethodsWe analyzed the oxylipin metabolome of paired tumors and adjacent normal mammary tissues from patients with pathologically confirmed breast cancer (N = 62). We used multivariate statistical analysis to identify important metabolite contributors and to determine the predictive power of tumor tissue metabolite clustering. In vitro functional assays using a panel of breast cancer cell lines were carried out to further confirm the crucial roles of endogenous and exogenous EETs in the metastasis transformation of TNBC cells. Deregulation of associated downstream signaling networks associated with EETs/CYPs was established using transcriptomics datasets from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Comparative TNBC proteomics using the same tissue specimens subjected to oxylipin metabolomics analysis was used as validation set.ResultsMetabolite-by-metabolite comparison, tumor immunoreactivity, and gene expression analyses showed that CYP epoxygenases and arachidonic acid-epoxygenation products, EET metabolites, are strongly associated with TNBC metastasis. Notably, all the 4 EET isomers (5,6-, 8,9-, 11,12-, and 14,15-EET) was observed to profoundly drive the metastasis transformation of mesenchymal-like TNBC cells among the TNBC (basal- and mesenchymal-like), HER2-overexpressing and luminal breast cancer cell lines examined. Our pathway analysis revealed that, in hormone-positive breast cancer subtype, CYP epoxygenase overexpression is more related to immune cell-associated signaling, while EET-mediated Myc, Ras, MAPK, EGFR, HIF-1α, and NOD1/2 signaling are the molecular vulnerabilities of metastatic CYP epoxygenase-overexpressing TNBC tumors.ConclusionsThis study suggests that categorizing breast tumors according to their EET metabolite ratio classifiers and CYP epoxygenase profiles may be useful for prognostic and therapeutic assessment. Modulation of CYP epoxygenase and EET-mediated signaling networks may offer an effective approach for personalized treatment of breast cancer, and may be an effective intervention option for metastatic TNBC patients.

BRCA1-associated R-loop affects transcription and differentiation in breast luminal epithelial cells.

BRCA1-associated basal-like breast cancer originates from luminal progenitor cells. Breast epithelial cells from cancer-free BRCA1 mutation carriers are defective in luminal differentiation. However, how BRCA1 deficiency leads to lineage-specific differentiation defect is not clear. BRCA1 is implicated in resolving R-loops, DNA-RNA hybrid structures associated with genome instability and transcriptional regulation. We recently showed that R-loops are preferentially accumulated in breast luminal epithelial cells of BRCA1 mutation carriers. Here, we interrogate the impact of a BRCA1 mutation-associated R-loop located in a putative transcriptional enhancer upstream of the ERα-encoding ESR1 gene. Genetic ablation confirms the relevance of this R-loop-containing region to enhancer-promoter interactions and transcriptional activation of the corresponding neighboring genes, including ESR1, CCDC170 and RMND1. BRCA1 knockdown in ERα+ luminal breast cancer cells increases intensity of this R-loop and reduces transcription of its neighboring genes. The deleterious effect of BRCA1 depletion on transcription is mitigated by ectopic expression of R-loop-removing RNase H1. Furthermore, RNase H1 overexpression in primary breast cells from BRCA1 mutation carriers results in a shift from luminal progenitor cells to mature luminal cells. Our findings suggest that BRCA1-dependent R-loop mitigation contributes to luminal cell-specific transcription and differentiation, which could in turn suppress BRCA1-associated tumorigenesis.

OR26-1 Phosphorylated Progesterone Receptor Isoforms Mediate Opposing Stem-Like and Proliferative Breast Cancer Cell Fates

Progesterone receptors (PRs) are key modifiers of ER target gene selection and drivers of luminal breast cancer progression. In addition to Her2 and estrogen receptor (ER), total PRs rather than isoform-specific PR expression levels are measured in breast tumors as indicators of functional ER. Herein, we identified phenotypic differences between PR-A and PR-B in luminal breast cancer cell models with focus on tumorsphere biology. Our findings indicate that PR-A is a dominant driver of cancer stem and stem-like cell (CSC) expansion in T47D breast cancer models, whereas PR-B is a potent driver of anchorage-independent proliferation. PR-A+ tumorspheres were enriched for ALDH activity, CD44+/CD24-, and CD49f+/CD24- (i.e. basal-like) cell populations relative to PR-B+ tumorspheres. Progestin promoted heightened expression of known CSC-associated target genes (e.g. FOXO1, p21, KLF4, WNT4, PTGES, NOTCH2) in PR-A+ but not PR-B+ cells cultured in non-adherent tumorsphere (3D) conditions. In contrast to previous reports, we report robust phosphorylation of PR-A relative to PR-B Ser294 (i.e. Ser130 in PR-A) and show that this residue is required for PR-A-induced expression of CSC-associated genes and CSC behavior in vitro. Chromatin immunoprecipitation assays demonstrated that PR-A phosphorylation was required for receptor recruitment to PRE-containing promoter regions of CSC-associated genes (WNT4, KLF4, NOTCH2). Remarkably, cells expressing S294A-PR-A exhibited severely impaired CSC phenotypes but heightened anchorage-independent cell proliferation. The PR target gene and co-activator, FOXO1, promoted PR Ser294 phosphorylation and tumorsphere formation. The FOXO1 inhibitor (AS1842856) alone or in combination with onapristone (PR antagonist) blunted phospho-PR (Ser294) and tumorsphere formation in PR-A+ and PR-B+ T47D cells and unmodified ER+/PR+ MCF-7 and BT474 breast cancer models. Our data reveal unique isoform-specific functions of phospho-PRs as modulators of distinct and opposing pathways (i.e. CSC expansion versus proliferation) in luminal breast cancer models. A clear understanding of PR isoform-specific actions, phosphorylation events, and the role of co-factors such as FOXO1 may lead to novel biomarkers of advanced tumor behavior and reveal new approaches to pharmacologically target CSCs in luminal breast cancers.

SUN-011 Identification Of Regulators Of Estrogenic Signalling In ER-positive Breast Cancer Cells By Whole Genome shRNA Screening

Over 70% of breast tumours express estrogen receptor alpha (ERα), a ligand-inducible transcription factor. Stimulation with estradiol leads to receptor binding to estrogen response elements (EREs) in target genes regulatory sequences in association with transcriptional cofactors. This results in altered gene expression, increased cell proliferation and accelerated tumour growth. To systematically identify genes contributing to ERα expression and signalling in breast cancer, we performed a genome-wide screen in human ERα+ luminal breast cancer cell lines stably expressing estrogen-induced luciferase reporters using an arrayed format. A primary screen (Sigma Mission lentiviral library, 16083 genes, three shRNA/gene) and secondary screens (615 genes identified as hits by at least two shRNAs with concordant impact on reporter gene expression without major effects on cell survival) were carried out using estrogen-sensitive cells (T47D-ERE3-Luc, MCF-7-ERE-Luc) and insensitive controls (antioxidant response reporter T47D-ARE-Luc). We ultimately characterized by RNASeq the impact of suppressing expression of each of 30 prioritized hits (2-3 shRNAs with >50% reduction of RNA levels per hit) on gene expression in ERα+ breast cancer cells. Knockdown of several of these hits had extensive impacts on expression of direct estrogen target genes and on ERα+ breast cancer cell proliferation. Some hits regulated ERα gene expression and several interacted with ERα in co-IP experiments, indicating roles as upstream regulators and/or transcriptional cofactors of ERα.

Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1\u03b1 gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1\u03b1 expression in breast cancer

HIF-1α has a broad impact on tumors, including enhanced utilization of glucose, tumor cell stemness, migration, metastasis and so on. In pilot study, we found that the expression of HIF-1α significantly increased in breast cancer cell lines and tissue samples with higher malignant behaviors and decreased in luminal subtype breast cancer cells and tissue samples. We analyzed and found there is one large CpG island in HIF-1α promoter around transcription start site, and the hypermethylation occurred at these CpGs and their surrounding non-CpGs sites. Epigenetic events driving tumorigenesis has been characterized. However, knowledge is lacking on the non-CpGs methylation of HIF-1α promoter in breast cancer cells. We validated that non-CpGs methylation can directly regulate HIF-1α expression by luciferase activity assay. We also found DNMT3a and Mecp2 play vital role in methylation at non-CpGs and CpGs sites. In addition, we noticed that H3K9ac modification could promote the transcription of HIF-1α in MDA-MB-231 cells by binding to the region contained hypomethylated non-CpG and CpG sites. Taken together, the hypomethylation status at non-CpG and CpG loci in HIF-1α promoter and H3K9ac modification together contribute to maintain higher HIF-1αactivity in invasive breast cancer cells when compared with the non-invasive breast cancer cells, which may establish a tissue-specific epigenetic modification pattern and point to the new directions for future understanding breast cancer therapy.

3300 Progesterone receptor alters lipid biology in luminal breast cancer

OBJECTIVES/SPECIFIC AIMS: These studies seek to evaluate hormonal regulation of luminal breast cancer lipid metabolism and to identify targetable progesterone-mediated changes in lipid biology that contribute to therapeutic resistance in breast cancer. METHODS/STUDY POPULATION: Established and patient-derived luminal breast cancer cell lines, which express ER and PR, were used for this study. RNA transcript and protein expression levels were evaluated by qRT-PCR and immunoblot, respectively. Broad scale lipidomics of progesterone-treated cells was conducted via ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) through the UCD Skaggs School of Pharmacy Mass Spectrometry Core. RESULTS/ANTICIPATED RESULTS: Data mining of previously published microarray data of CK5+ and CK5− syngeneic cancer sublines revealed that CK5+ cells have increased expression of lipid processing genes, including LPL and PPARG. As progestin treatment induces a subpopulation of cells to turn on CK5 expression in luminal breast cancers, UHPLC-MS-based lipidomics analysis will expose whether modulation of the lipid landscape occurs in all cells with progesterone treatment, or whether this phenomenon is heightened specifically in CK5+ cells. I also expect that ER+ breast cancers with progestin induced-altered lipid content, such as lipid droplet formation, will evade therapy-induced death. DISCUSSION/SIGNIFICANCE OF IMPACT: There are numerous approved and developmental therapeutics targeting lipid biology. By determining if progestins alter lipid metabolic genes specifically in CK5+ CSCs, which are endocrine resistant, strategies may be devised to target these resistant cells using combination therapy in conjunction with existing therapies to prevent tumor recurrence.

TNF‑α inhibits xenograft tumor formation by A549 lung cancer cells in nude mice via the HIF‑1α/VASP signaling pathway.

Lung cancer is the leading cause of cancer‑associated mortality worldwide. Tumor necrosis factor α (TNF‑α) is an important cytokine in the tumor microenvironment that serves a function in the balance of cell survival and cell death pathways. Our previous studies indicated that hypoxia‑inducible factor 1α (HIF‑1α) acts downstream of TNF‑α in MCF‑7 luminal breast cancer cells. However, whether vasodilator‑stimulated phosphoprotein (VASP) is implicated in the direct regulation of HIF‑1α in response to TNF‑α in lung cancer remains unknown. In vitro studies were performed using A549 and H226 lung carcinoma cells and in vivo studies of tumor xenograft models were performed to investigate the effects of TNF‑α. The results demonstrated that TNF‑α decreased VASP expression by upregulating the expression of HIF‑1α to inhibit A549 cell proliferation and adhesion. Inhibition of transplanted tumor growth was associated with downregulation of VASP expression in nude mice. Bioinformatics analysis indicated that expression levels of VASP or HIF‑1α lead to differential outcomes of overall survival in lung carcinoma. These results suggest that the HIF‑1α/VASP signaling pathway serves an important function in the regulation of TNF‑α‑induced suppression of A549 cell proliferation and xenograft growth. This may improve our understanding of the antitumor effect of TNF‑α.

Abstract P5-04-24: Molecular stratification of ER+/HER2- breast cancer cell lines to predict sensitivity to targeted agents

Abstract Background: Approximately 70% of all breast cancers are estrogen receptor positive (ER+) at diagnosis and are dependent on estrogen signaling for tumour growth and proliferation. Some ER+ breast cancers can be effectively treated with adjuvant endocrine therapies including tamoxifen, but despite favorable improvements in overall survival, resistance to endocrine therapy is common and has been associated with dysregulation of several signaling pathways. These pathways can be targeted with specific inhibitors, many of which are currently under clinical investigation. However currently there is a lack of predictive biomarkers to identify which patients should receive treatment with targeted therapy. The goal of this study was to determine whether alterations in specific signaling pathways can be identified and used to stratify breast cancer cell lines to the most effective experimental treatments. Methods/Results: Fifteen ER+/HER2- cell lines were characterized using a NanoString PAM50-like assay as well as next generation sequencing and were then stratified according to alterations in three key signaling pathways: CCND/CDK, PI3K/AKT/mTOR and FGFR. High-throughput small-molecule screenings were performed to identify the IC50 values of 24 inhibitors across the strata. Variation in inhibitor sensitivity was observed between cell lines based on molecular alterations. Cell lines with a PIK3CA mutation in combination with a CDK-pathway alteration were more sensitive to CDK inhibitors (50 to 120nM) than cell lines with alterations in the CDK-pathway alone or PIK3CA mutations alone (170nM to >5000nM). In addition, cell lines with the dual alterations demonstrated stronger synergy between CDK and PI3K-pathway inhibitors compared to either alteration alone. Conclusions: The results suggest that stratification according to molecular alterations in specific signaling pathways may predict sensitivity to targeted inhibitors in a panel of ER+/HER2- luminal breast cancer cell lines. Work is ongoing to identify the optimal synergistic inhibitor combinations for each strata. The ultimate goal is to translate this work into a novel personalized medicine approach, using molecular stratification based on a combination of molecular events in a functional pathway as opposed to single genes. Citation Format: Bathurst L, Liao L, Crozier C, Lyttle N, Marcellus R, Bayani J, Al-awar R, Bartlett J, Spears M. Molecular stratification of ER+/HER2- breast cancer cell lines to predict sensitivity to targeted agents [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-24.

Abstract P4-01-24: S100A11 is a diagnostic marker and promotes cancer progression for luminal breast cancer

Abstract Background: S100A11, also known as calgizzarin, is a member of S100 calcium-binding protein family and is upregulated in various cancers including lung cancer, renal cell cancer, ovarian cancer and pancreatic cancer. Many studies have shown that S100A11 is related to cancer progression. However, the function of S100A11 in breast cancer remains largely unknown. In the present study, we aim to investigate the underlying mechanism(s) of S100A11 in cancer cell proliferation, invasion, migration, and evaluate the clinical association with breast cancer. Methods: The mRNA expression level of S100A11 in plasma samples from breast cancer patients and normal control individuals was detected with quantitative real-time PCR. Knockdown of S100A11 using small interfering RNA (siRNA) in luminal breast cancer cells (MCF-7 and T-47D) were used to study the biological function of S100A11 for the progression of breast cancer. Cell proliferation ability was analysed with MTT assay and colony formation assay. Cell invasion ability was analysed with transwell invasion assay. Cell migration ability was detected by wound healing assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Results: The mRNA expression level of S100A11 was significantly increased in the plasma samples obtained from 182 breast cancer patients compared with 115 normal control individuals, and the area under curve was 0.83. In particular, higher expression was associated with luminal breast cancer subtype as well as respective cell lines. S100A11 siRNA effectively inhibited the proliferation, invasion, and migration abilities of MCF-7 and T-47D cells. Knockdown of S100A11 caused cell cycle G1 arrest and induced apoptosis. Silencing of S100A11 decreased the mRNA and protein expression levels of cyclin D1 and NF-κB p50 whereas increased the expression level of E-cadherin in MCF-7 and T-47D cells. Conclusion: These results suggest that S100A11 is upregulated in breast cancer and promoted cell proliferation, invasion and migration in MCF-7 and T-47D cells through the upregulation of NF-κB p50 and cyclin D1. Thus, S100A11 may be a potential diagnostic marker for luminal breast cancer subtype and also a therapeutic target for breast cancer. Citation Format: Kwong A, Wang J, Chen J, Siu MT, Chuek I, Shin V. S100A11 is a diagnostic marker and promotes cancer progression for luminal breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-24.

Abstract P5-05-06: Progesterone receptor (PR) isoform-specific expansion of breast cancer stem-like cells occurs through a phospho-PR/FOXO1 driven signaling axis

Abstract Luminal breast cancers account for ˜75% of cases and are positive for progesterone receptor (PR) and estrogen receptor (ER) expression. PR is a classical ER-target gene and is used as a biomarker of ER activity; however, a growing body of evidence supports the role of PR as an important modifier of ER actions and a key driver of luminal breast cancer progression. Progesterone signaling is mediated by two PR isoforms: full-length PR-B and truncated PR-A, which lacks the N-terminal 164 amino acids. Little is known about PR isoform-specific actions in PR-expressing breast tumors, given that total PR expression is measured clinically. Herein, we sought to identify phenotypic differences in luminal breast cancer cells (T47D) overexpressing PR-A (T47D-YA) or PR-B (T47D-YB). We demonstrate that PR-B expression is required for anchorage-independent colony formation, while PR-A expressing cells fail to proliferate in soft agar. PR-B driven proliferation has been mapped to PR phosphorylation events, which include MAPK or CDK consensus sites such as Ser294. We demonstrate that in contrast to previous reports, PR-A is well phosphorylated at Ser294 in response to progestins (e.g. R5020) using our custom phospho-PR (Ser294) polyclonal antibody. Interestingly, Ser294 phosphorylation of PR-A occurs more rapidly and robustly following hormone treatment compared to PR-B expressing cells. Our findings indicate that PR-A is a dominant driver of stem-like expansion in breast cancer cells. PR-A tumorspheres exhibited enriched ALDH+ and CD44+/CD24- populations compared to PR-B and promoted heightened gene expression of stem-like genes (e.g. FOXO1). We demonstrate that the PR target gene and co-activator FOXO1 promotes both PR-A and PR-B phosphorylation at Ser294 and augments tumorsphere formation. Direct inhibition of FOXO1 levels abrogates phospho-PR (Ser294) levels and tumorsphere formation in PR expressing cells. Finally, we show that Ser294 is required for PR-A induced expression of stem-like genes (e.g. FOXO1) and stem-like behavior as measured by ALDH+/CD44+ tumorspheres. Taken together, our data reveal unique functions of PR isoforms as modulators of distinct and opposing pathways (i.e. proliferation versus stem-like expansion) in luminal breast cancer models. A clear understanding of PR isoform-specific actions, phosphorylation events, and the role of co-factors such as FOXO1 may lead to novel biomarkers of advanced tumor behavior and reveal new approaches to pharmacologically target PR isoforms in luminal breast cancers. Citation Format: Truong TH, Dwyer AR, Diep CH, Lange CA. Progesterone receptor (PR) isoform-specific expansion of breast cancer stem-like cells occurs through a phospho-PR/FOXO1 driven signaling axis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-06.

Abstract P2-02-15: All-trans retinoic acid perturbs the lipidomic profiles of luminal breast cancer cells characterized by sensitivity to the anti-proliferative activity of the retinoid

Abstract Background: All-trans retinoic acid (ATRA) is the active metabolite of vitamin A and a promising agent in the prevention and treatment of breast cancer. We recently demonstrated that approximately 70% of estrogen-receptor-positive (ER+) breast cancer cell lines and primary tumors are sensitive to the anti-proliferative effects of ATRA (1,2). In contrast only 10-20% of the HER2-positive and triple-negative counterparts respond to the retinoid. The significance of lipids in the growth, progression and drug sensitivity of specific types of solid tumors, including breast cancer, is largely overlooked. In particular the role, if any, of specific lipids in the anti-tumor action of ATRA in breast cancer has never been studied. Aims and Approach: The principal aim of the present work was to evaluate the specific perturbations induced by ATRA on the homeostasis of lipids in breast cancer cells characterized by sensitivity to the anti-proliferative action of the retinoid. To this purpose, we took a high-throughput approach and defined the lipidomic profiles of 16 breast cancer cell lines in basal conditions and following challenge with ATRA (1 μM) for 48 hours. The panel consisted of eight cell lines characterized by a luminal phenotype and eight cell lines with a basal phenotype. Four ATRA-sensitive cell lines (SKBR3, HCC1500, CAMA1 and MDAMB361) and 4 ATRA-resistant counterparts (HCC202, MDAMB175VII, ZR75.1 and HCC1419) were included in the luminal group. Similarly, the basal group contained 4 ATRA-sensitive (HCC1599, MB157, MDAMB157 and Hs578T) and 4 ATRA-resistant (MDAMB231, CAL851, HCC1187 and MDAMB436) cell types. Results: Using Lipostar, a unique and recently developed software for high-throughput LC-MS lipidomics analysis (3), we identified lipid species whose levels were modified by ATRA in each cell line. This resulted in the generation of a lipid fingerprint consisting of 530 elements. We observed that ATRA reduced the amounts of cardiolipins in luminal and ATRA-sensitive breast cancer cell lines specifically. Similar effects were not observed in luminal and ATRA-resistant cells. ATRA-dependent reduction in the amounts of cardiolipins were never observed in basal cells, regardless of their sensitivity to the retinoid. Given the role played by cardiolipins in the homeostasis of the mitochondria, we evaluated the action of ATRA on the functional activity of these organelles in the luminal and ATRA-sensitive or ATRA-resistant cell lines. In SKBR3 and the other sensitive cell lines, we observed that ATRA modulated mitochondria-dependent oxygen consumption and ATP production. These effects were accompanied by an increase in mitochondrial membrane fluidity, which is consistent with the effects exerted by ATRA on the cellular content of cardiolipins. ATRA-dependent action on cardiolipins and mitochondrial homeostasis precedes maximal growth inhibition. The results provide new insights on the mechanisms underlying the anti-tumor action of ATRA. References : 1) Centritto F. et al. EMBO Mol Med. 2015 Jul;7(7):950-72. 2) Bolis M et al.. Ann Oncol. 2017 Mar 1;28(3):611-621. 3) Goracci, L. et al., Anal Chem 2017, 89 (11), 6258-6265. Citation Format: Terao M, Celestini V, Kurosaki M, Vallerga A, Bolis M, Fratelli M, Paroni G, Di Veroli A, Cruciani G, Goracci L, Garattini E. All-trans retinoic acid perturbs the lipidomic profiles of luminal breast cancer cells characterized by sensitivity to the anti-proliferative activity of the retinoid [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-15.

Abstract P5-04-28: A potential role for IL6ST mediating endocrine resistance in breast cancer via interaction with the ER signaling pathway

Abstract Background Interest in the ubiquitously expressed transmembrane receptor IL6ST (gp130) has developed as it has been identified as a predictive biomarker of endocrine treatment response in breast cancer patients and is included in the 'Endopredict' test. At least seven cytokines (IL-6, OSM, LIF, IL-11, CNTF, IL-27, CT-1) signal via IL6ST. Interleukin-6 (IL-6) can mediate effects via two signaling pathways; classic signaling (through the membrane-bound IL-6 receptor, IL-6R) and trans-signaling (via non-signaling membrane-bound soluble IL-6R, sIL-6R). Both pathways may occur in parallel and activate cells. Crosstalk between the ER signaling pathway and IL6ST through STAT3 has been identified and implicated in the conversion from ER responsiveness to non-responsiveness. Method A panel of 3 ERa+ luminal breast cancer cell lines (MCF-7, T47D, ZR-75-1) were chosen to examine IL6ST expression by western blot, gel electrophoresis and qRT-PCR. Proliferation assays (SRB) were carried out to investigate the effects of seven cytokines (IL-6, OSM, LIF, IL-11, CNTF, IL-27, CT-1) on cell growth. The action of both IL-6 and Oncostatin M (OSM) on cell migration and downstream signaling pathways (pSTAT3 and pERK1/2) was studied. The extent of trans-signaling occurrence and interaction between cytokines and estrogen was investigated using proliferation assays. Results Three cell lines (MCF-7, T47D, ZR-75-1 were shown to express varying levels of full-length IL6ST, with MCF-7 having the least expression. Gel electrophoresis and qRT-PCR confirmed the presence of all previously described IL6ST soluble forms in the three cell lines. Surprisingly, the growth response to the cytokines was variable across the cell lines. IL6 caused a modest increase in growth in MCF-7 but produced inhibition in ZR-75-1. OSM and LIF stimulated growth in MCF-7, whereas OSM inhibited ZR-75-1 and LIF had no effect. Interestingly, no significant effect on growth was seen in T47D with any of the cytokines except IL-11 which generated a significant growth effect on T47D cells. Both STAT3 and MAPK/ERK pathways were activated to different extents in the three cell lines as a result of OSM and IL-6 activation, whereas cells migration was only stimulated in ZR-75-1. The trans-signaling pathway significantly enhanced the growth inhibition in ZR-75-1. Estrogen eliminated the effect of both IL-6 and OSM on MCF-7 growth, while both cytokines decreased estrogen-induced cell proliferation in ZR-75-1. There was no effect in T47D. Conclusion These results indicate: · The presence of both classic and trans-signaling occurrence in breast cancer cell lines. · Differential patterns of pSTAT3 and pERK1/2 signaling which could help explain the variation in responses to the cytokines. · IL6ST full-length form is the most abundant form in all three cell lines under basal conditions. · The levels and roles of the different IL6ST soluble forms will be further studied after estrogen stimulation. · The effect of IL6ST silencing in the presence of estrogen and tamoxifen on cell growth and the gene expression currently being examined. Citation Format: Mosly DH, Turnbull AK, Langdon SP, Sims AH. A potential role for IL6ST mediating endocrine resistance in breast cancer via interaction with the ER signaling pathway [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-28.

Abstract P2-01-11: A peptide with the conserved amino acid residue of integrin α6 inhibits metastasis through disruption of complex formation in breast cancer cells

Abstract Metastasis inhibition may improve survival of breast cancer patients. Our previous study revealed that a transcription factor SALL4 promotes cell migration for metastasis through up-regulation of integrin α6β1 in metastatic basal-like breast cancer cells. In these cells, integrin α6β1 modulates Rho GTPase activity, which enhances focal adhesion dynamics for cell migration. We therefore focused on integrin α6 as a therapeutic target for metastasis. We investigated the expression level of integrin α6 in breast cancer cell lines and a tissue microarray of breast cancer patients. In both experiments, we observed higher integrin α6 expression in basal-like breast cancer cells than in luminal ones. We performed shRNA-mediated integrin α6 knockdown in basal-like breast cancer cells and conducted Boyden chamber assays. In the results, reduced cell migration was observed in integrin α6 knocked-down cells. Reciprocally, we introduced integrin α6 overexpression in low-migratory luminal breast cancer cell lines and observed increased cell migration. These results indicate that integrin α6 promotes cell migration in breast cancer cells. To identify the functional residue of integrin α6, we analyzed its amino acid sequence by in silico analyses. First, we extracted integrin α6 specific residues in integrin α family in the human genome. Then, we compared integrin α6 sequences among vertebrates. In the result, we obtained an integrin α6-specific and vertebrate-conserved sequence, Asp-358, as a candidate for the functional residue. To inhibit integrin α6 function, we designed an 8-amino acid peptide with the sequence around Asp-358. Basal-like breast cancer cells treated with the peptide showed reduced cell migration in a dose-dependent manner. The peptide did not reduce cell migration in integrin α6 knocked-down cells, suggesting that the peptide inhibits integrin α6 function. To determine whether the peptide inhibits metastasis, we performed zebrafish metastasis assays, and observed reduced metastasis rate in the peptide-treated group. These results indicate that the peptide inhibits metastasis through reduction in cell migration. To understand the effect of the peptide, we performed chemical cross-liking assay in a basal-like breast cancer cell line. Protein samples from the cells treated with a cross-linker showed bands of integrin α6 complexes, and the intensities of these bands were reduced in the peptide-treated group. Moreover, we immunostained cells with an antibody for focal adhesion marker, phospho-paxillin, because integrins regulates focal adhesion formation for cell migration. In the results, the peptide reduced the number of focal adhesions. These observations indicate that the peptide inhibits integrin α6 function. In the future, our findings may contribute to development of a metastasis inhibition therapy. Citation Format: Itou J, Tanaka S, Senda N, Iida A, Sehara-Fujisawa A, Sato F, Toi M. A peptide with the conserved amino acid residue of integrin α6 inhibits metastasis through disruption of complex formation in breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-11.

Targets and molecular mechanisms of a citrus flavonoid, hesperidin, against luminal breast cancer cells: an integrative bioinformatics analysis

Objective: To identify the potential target and mechanisms of hesperidin in MCF-7 estrogen receptor-positive breast cancer cells using bioinformatics approaches. Methods: Gene expression profiles were accessed from public database GSE85871. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was carried out with Database for Annotation, Visualization and Integrated Discovery. The protein-protein interaction network was analyzed by STRING-DB and visualized by Cytoscape. Transcription factor regulatory networks were constructed from TRED, TRRUST, RegNetwork and visualized by Cytoscape. Drug association analysis was conducted by WebGestalt. Results: GO and KEGG pathway enrichment analysis revealed biological processes, cellular components and molecular functions that were related to cancer and estrogen signaling pathways. KEGG pathway enrichment analysis of the genes in transcription factor-differential expression genes regulatory network showed regulation of cancer, estrogen signaling pathways, epidermal growth factor receptor tyrosine kinase inhibitor resistance, and endocrine resistance. Moreover, drug association analysis revealed that hesperidin affected the expression of the same gene as raloxifene. Conclusions: Hesperidin targets estrogen receptor signaling in estrogen receptor-positive breast cancer cells. Results of this study could trace the molecular mechanism of hesperidin in estrogen receptor-positive breast cancer cells and integrative bioinformatics analysis could accelerate drug discovery and development.

Targeting the BRD4/FOXO3a/CDK6 axis sensitizes AKT inhibition in luminal breast cancer

BRD4 assembles transcriptional machinery at gene super-enhancer regions and governs the expression of genes that are critical for cancer progression. However, it remains unclear whether BRD4-mediated gene transcription is required for tumor cells to develop drug resistance. Our data show that prolonged treatment of luminal breast cancer cells with AKT inhibitors induces FOXO3a dephosphorylation, nuclear translocation, and disrupts its association with SirT6, eventually leading to FOXO3a acetylation as well as BRD4 recognition. Acetylated FOXO3a recognizes the BD2 domain of BRD4, recruits the BRD4/RNAPII complex to the CDK6 gene promoter, and induces its transcription. Pharmacological inhibition of either BRD4/FOXO3a association or CDK6 significantly overcomes the resistance of luminal breast cancer cells to AKT inhibitors in vitro and in vivo. Our study reports the involvement of BRD4/FOXO3a/CDK6 axis in AKTi resistance and provides potential therapeutic strategies for treating resistant breast cancer.