bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P6-02-08: Molecular drivers of adipogenotoxicosis in breast tumor-associated adipose
Authors: Ellsworth RE , Field LA , van Laar R , Deyarmin B , Hooke JA , Shriver CD
Published: August 2014

Background: Having long been thought to function only as an inert energy storage depot, the role of adipose tissue in tumorigenesis has been largely ignored; however, adipose is an active endocrine organ that can directly influence tumor growth. Improved understanding of the role of adipose in tumorigenesis is crucial given the association between obesity and breast cancer risk in post-menopausal women, increasing rates of obesity and use of autologous fat transfer in breast reconstruction.

Methods: Adipose, adjacent to and distant from (>3 cm from the closest tumor margin) invasive breast tumors, was laser microdissected from 20 post-menopausal women, and from 22 post-menopausal women with non-malignant breast disease. Gene expression data were generated using U133A 2.0 microarrays. Data were analyzed to identify significant patterns of differential expression between adipose classes at the individual gene and molecular pathway level. Gene expression differences were validated using qRT-PCR in an additional set of 29 specimens.

Results: SPP1, RRM2, MMP9 and PLA2G7 were expressed at >3-fold (P < 0.01) higher levels in adjacent adipose compared to distant adipose from the same breast. A number of immune response genes including MARCO, FABP7, ELF5, MYBPC1, MMP7, CLDN8, HLA-DQB1 and HLA-DQA1 were differentially expressed in distant adipose compared to adipose from non-malignant breasts. The most significant gene expression differences were detected between tumor-adjacent and non-malignant adipose with >3-fold higher expression of EGFL6 and ITGB2 and >3-fold lower levels of PIP, which are involved in growth, proliferation, and cellular adhesion in adjacent compared to non-malignant adipose. Pathway analysis revealed that immune response differs between non-malignant, distant and tumor-adjacent adipose with an enhanced B- and T-cell response detected in adjacent compared to distant or non-malignant adipose. Inflammatory response as well as DNA transcription and replication pathways were differentially expressed in distant compared to non-malignant adipose.

Conclusions: Gene expression levels differ in breast adipose depending on presence of and proximity to tumor cells. Adipose adjacent to the tumor demonstrated the largest immune response, supporting the idea of adipogenotoxicosis, which through pro-inflammatory and genotoxic responses, promotes tumor development. In addition, genes involved in cellular proliferation, degradation of the extracellular matrix and angiogenesis are differentially expressed in adjacent compared to distant or non-malignant adipose, thus tumor-adjacent adipose may be contributing to the growth and invasion of the primary tumor. These data thus suggest that adipose is not an inert component of the breast microenvironment but plays an active role in tumorigenesis.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-08.
Abstract P6-02-09: Role of HGF in obesity-associated tumorigenesis: C3(1)-Tag mice as a model for human basal-like breast cancer
Authors: Sundaram S , Freemerman AJ , Hamilton J , McNaughton K , Galanko JA , Darr DB , Perou CM , Troester MA , Makowski L
Published: August 2014

Obesity is associated with basal-like breast cancer (BBC), an aggressive breast cancer subtype. The objective of this study was to determine whether high fat diet-induced obesity promotes BBC onset in adulthood and to evaluate the role of stromal-epithelial interactions in obesity-associated tumorigenesis. Specifically, we hypothesized that hepatocyte growth factor (HGF) plays a promoting role in BBC, which tends to express the HGF receptor, c-Met. In C3(1)-Tag mice, a murine model of BBC, we demonstrate that obesity leads to a significant increase in HGF secretion and an associated decrease in tumor latency. By immunohistochemical analysis, normal mammary tissue exhibit obesity-induced hepatocyte growth factor (HGF) in stroma and corresponding epithelial expression of c-Met. HGF secretion was also increased in primary mammary fibroblasts isolated from normal mammary and tumors of obese mice compared to lean. These results show that diet-induced elevation of HGF expression is a stable phenotype, maintained after several passages and after removal of dietary stimulation. In cocultures, neutralization of HGF blunted tumor cell migration; further linking diet-mediated HGF-dependent effects in tumor aggressiveness. In sum, these results suggest that HGF plays an important role in obesity-associated carcinogenesis and raise the hypothesis that diet may affect lasting changes in stroma, potentially through epigenetic means. Understanding the effects of obesity on risk and progression is important given epidemiologic studies that imply half of BBC could be eliminated by reducing obesity. Our findings suggest the possibility that reducing obesity may be useful in preventing obesity-associated breast cancer mediated by the HGF/c-Met pathway.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-09.
Abstract P6-04-02: Final progression-free survival analysis of BOLERO-2: a phase III trial of everolimus for postmenopausal women with advanced breast cancer
Authors: Piccart M , Baselga J , Noguchi S , Burris H , Gnant M , Hortobagyi G , Mukhopadhyay P , Taran T , Sahmoud T , Rugo H
Published: August 2014

Introduction: Treatment options for postmenopausal women with hormone receptor-positive (HR+) breast cancer (BC) who relapse or progress on a nonsteroidal aromatase inhibitor (NSAI) are limited. Interim analyses of the BOLERO-2 trial demonstrated that combining the oral mammalian target of rapamycin (mTOR) inhibitor, everolimus (EVE), with the steroidal aromatase inhibitor, exemestane (EXE), significantly prolonged progression-free survival (PFS) in this patient population. Protocol-specified final PFS analyses are presented.

Methods: The phase III, double-blind, BOLERO-2 trial randomized postmenopausal women with HR+ BC progressing or recurring after NSAIs to EVE (10 mg once daily; n = 485) plus EXE (25 mg once daily) or placebo (PBO; n = 239) plus EXE. The primary endpoint was PFS. Secondary endpoints included overall survival, safety, bone turnover, and overall response rate.

Results: At a median follow-up of 18 months, 510 PFS events were reported. The addition of EVE to EXE significantly prolonged median PFS versus EXE monotherapy as per local assessment (7.8 vs 3.2 mo, respectively; HR = 0.45 [95% CI, 0.38–0.54]; log-rank P < .0001). Furthermore, these data were consistent with results based on central assessment (11.0 mo for EVE + EXE vs 4.1 mo for EXE alone; HR = 0.38 [95% CI, 0.31–0.48]; log-rank P < .0001). Fewer deaths were reported with EVE + EXE (25.4%) vs PBO + EXE (32.2%). In addition to significantly improving PFS and delaying disease progression in bone, PFS benefits with EVE + EXE versus PBO + EXE were consistent in all prospectively defined subgroups, including subsets of patients with visceral metastases (n = 406), 3 or more sites of metastasis (n = 271), and Eastern Cooperative Oncology Group (ECOG) performance status 1 or 2 (n = 274). The safety profile of EVE + EXE was consistent with that reported at the interim analysis and with data for EVE from other oncology settings. Updated overall survival data will be presented.

Conclusions: Adding EVE to EXE markedly prolonged PFS in the overall population and in all patient subsets, validating the rationale of targeting the mTOR pathway to improve clinical outcome in patients with HR+ advanced BC progressing during or after NSAI therapy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-02.
Abstract P6-04-03: Changes in breast tumor metabolism and estradiol binding as measured by FES PET in patients treated with the histone deacetylace inhibitor vorinostat and aromatase inhibitor therapy
Authors: Linden HM , Kurland BF , Specht JM , Vijayakrishn GK , Gralow JR , Peterson LM , Schubert EK , Link JM , David MA , Eary JF , Krohn KA
Published: August 2014

Background: Some estrogen receptor-positive (ER+) metastatic breast cancers are bone and soft tissue dominant, indolent, and controlled by endocrine therapy. However, these tumors eventually become refractory to endocrine therapy and need a mechanism to reset the “estrogen-dependence” to allow continued benefit upon progression. Histone deacetylase inhibitors (HDACi) act as modulators of gene expression that are promising therapeutic agents for this group of tumors (Huang 2000, Sabnis 2011). Preclinical and clinical data demonstrate in ER-poor tumors and cell lines ER up-regulation and consequently enhanced lethality to endocrine agents. The optimal dose and schedule are not known, but two promising phase II studies show benefit in a continuous schedule (Yardley 2011, Munster 2011). FES PET is a promising imaging agent used as a biomarker to determine which patients will benefit from endocrine therapy, and to monitor estradiol binding during therapy (Mortimer 2001, Linden 2011).

Methods: Patients with ER+ HER2− metastatic breast cancer with prior aromatase inhibitor (AI) exposure and clinical benefit of endocrine therapy were eligible for a phase II study of HDACi therapy to restore sensitivity to AI therapy. Following baseline FDG PET, FES PET and standard imaging (CT, MRI, ultrasound and/or bone scan as indicated by tumor location), patients received 2 weeks of vorinostat therapy (400 mg po daily). FES PET was performed at 2 weeks while on HDACi therapy. Patients then received 6 weeks of AI monotherapy. FDG PET, FES PET and response assessment were performed at 8 weeks. Patients with clinical benefit (stable disease or response) continued on the regimen, 2 weeks of vorinostat followed by 6 weeks of AI.

Results: To date, 8 patients have been enrolled of whom 6 have completed the first 8 weeks of treatment and all correlative imaging studies. FES biomarker imaging results are mixed, with some patients showing an increase in tumor estradiol concentrating ability by FES PET on HDACi therapy, and decline in metabolic activity by FDG. Two patients continue on treatment with clinical benefit. Results will be updated as accrual continues.

Conclusions: Changes in estradiol binding are measured by serial FES PET in patients on HDACi therapy support preclinical concept of HDACi modulation of ER expression in metastatic breast cancer. Molecular imaging is a promising tool to monitor Estradiol binding pharmacodynamics, and Vorinostat HDACi therapy is a promising novel approach to allow patients to avoid toxicities of traditional chemotherapy once their tumor has progressed on endocrine therapy.

Funding: P01, MKA, Merck

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-03.
Abstract P6-04-11: Combination of PI3K-AKT-mTOR and MEK-ERK pathway inhibitors overcome acquired resistance to letrozole in ER+ breast cancer models
Authors: De P , Sun Y , Friedman LS , Chen S , Dey N , Leyland-Jones B
Published: August 2014

Background: Approximately 60–70% of breast cancers (BC) express estrogen receptor (ERα) and/or progesterone receptor (PR), the most common malignancy in women in the US and Europe. Many though not all patients with ER+ BC respond to hormonal therapy (e.g. tamoxifen and aromatase-inhibitor) but a large number will eventually develop resistance to long term therapy.

Purpose: Development of resistance to endocrine therapy is a clinical issue in ER+ BC. Studies with human cell lines have shown that upregulation of PI3K-AKT-mTOR and MEK-ERK pathways contributes to resistance to endocrine therapy (Miller TW et al JCO 2011; Sanchez CG et al Breast Cancer Res 2011). Using leterozole-resistant ER+ BC model, we investigated the effects of PI3K-AKT-mTOR pathway inhibitor alone and in combination with MEK1/2 inhibitor.

Methodology: The anti-proliferative, anti-migratory, and intracellular signaling (pAKT, pS6RP, p4EBP1 and pERK) effects of GDC-0941 (Class I PI3K inhibitor) or GDC-0980 (dual PI3K/mTOR inhibitor) alone and in combination with GDC-0973 (MEK1/2 inhibitor) were evaluated in aromatase overexpressed (MCF-7aro), letrozole-resistant (MCF-7aro/LetR) and long-term-estrogen-deprived (LTEDaro) BC cell lines by MTT assays, integrin-mediated migration assay and Western blots.

Results: 1) Both GDC-0980 and GDC-0941 exhibited excellent in vitro cell killing activity in MTT assay in all cell lines with IC50's 10–30 nM & 0.2–1.5 µM respectively. PI3K pathway inhibitors were more potent when combined with GDC-0973; 2) Integrin-mediated migration was significantly higher in both MCF-7aro/LetR andLTEDaro cells than MCF-7 and MCF-7aro cells; 3) Both MCF-7aro/LetR and LTEDaro cells movement were significantly abrogated following the treatment of GDC-0980, and the combination of GDC-0980 plus GDC-0973 more effectively blocked integrin-mediated migration; 4) Inhibition of phosphorylation of AKT (S473/T308), S6RP and 4EBP1 (T37/46) was observed following PI3K pathway inhibitors; 5) pERK was upregulated following the treatment of GDC-0941 (PI3K inhibitor); 6) Basal level of pERK was significantly high in MCF7/LetR and MCF7/LTED cells, and GDC-0980 failed to change the level of pERK in those cell lines; and 7) Combination of PI3K pathway inhibitor plus MEK1/2 inhibitor significantly blocked activation of both pathways.

Conclusion: Our in vitro data suggest that therapeutic targeting of the PI3K-AKT-mTOR and the MEK-ERK signaling pathways should be effective in abrogating aromatase-inhibitor-resistance in ER+ BC.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-11.
Abstract P6-04-18: Evaluation of the molecular mechanisms behind fulvestrant resistant breast cancer
Authors: Kirkegaard T , Hansen SK , Reiter BE , Sorensen BS , Lykkesfeldt AE
Published: August 2014

To study the mechanisms behind treatment resistance, we have previously established a large panel of antiestrogen resistant breast cancer cell lines based on the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. It seems to be a general phenomenon that MCF-7 cells switch from ER driven to human epidermal growth factor receptor (HER) driven cell growth upon development of fulvestrant resistance. To obtain a broader knowledge of the resistance mechanisms, we have established four fulvestrant resistant subclones from the ER-positive breast cancer cell line, T47D.

The parental T47D cells were confirmed to be estrogen responsive and short-term treatment of the parental T47D cells with fulvestrant severely reduced cell growth as well as protein expression of ER and the estrogen regulated proteins, insulin-like growth factor 1 receptor alpha (IGF-1Rα) and progesterone receptor (PR). All four fulvestrant resistant cell lines had reduced mRNA and protein expression of HER1 and HER4 compared to T47D, while HER2 was highly up regulated in the fulvestrant resistant cell lines and ER was lost. No difference in response to the HER2 inhibitor trastuzumab or the specific HER2 kinase inhibitor AG825 was observed between the fulvestrant resistant cell lines and T47D, indicating that HER2 is not the main driver of fulvestrant resistance in T47D cells. We found no indication of involvement of the two classical signaling pathways downstream of the HER receptors (Ras/Raf/Erk and PI3K/Akt). However, the JNK inhibitor SP600125 preferentially inhibited growth of two tested fulvestrant resistant cell lines. Withdrawal of fulvestrant for several weeks did not result in regain of ER expression, or in change of HER receptor expression, supporting a stable resistant phenotype.

In conclusion, loss of ER expression explains the lack of effect of fulvestrant in the T47D fulvestrant resistant cell lines. Noteworthy, the observed over expression of HER2 in the fulvestrant resistant T47D cells does not seem to be the main driver of cell growth, as HER2 targeted therapies like trastuzumab and HER2 kinase inhibitor did not exert a preferential growth inhibition of resistant cells. These breast cancer cells with high endogenous HER2 expression will be used as a model mimicking HER2 positive breast cancer which does not respond to standard HER2 targeted therapies.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-18.
Abstract P6-04-22: Regulation of Notch localization by endocrine therapy in Estrogen Receptor positive breast cancer cells: Clinical implications for endocrine resistance
Authors: Espinoza I , Caskey M , Baker RC , Miele L
Published: August 2014

Background: Estrogen receptors occur in about two-thirds of breast tumors and endocrine therapy is probably the most important systemic therapy for hormone receptor positive breast cancer. However, after an initial response to hormonal therapy, most tumors develop resistance leading to disease progression. Central mechanisms thought to be involved in this process include: 1) alterations in tumor cell physiology causing insensitivity to drug-induced apoptosis or induction of drug-detoxifying mechanisms; 2) expression of energy-dependent transporters that eject anti-cancer drugs from cells and 3) the emergence of cancer stem-like cell clones that are estrogen-independent and/or antiestrogen resistant. We and others have shown that Notch signaling mediates survival signals in ER+ breast cancer cells; induces expression of drug transporter ABCG2 (BCRP), activates ERα in the absence of estrogen and mediates survival of breast cancer stem-like cells. Previously, we demonstrated a crosstalk between ERα and Notch signaling whereby estrogen inhibits Notch activation and estrogen withdrawal or tamoxifen re-activate Notch, a possible mechanism of resistance. The mechanism of these effects has remained elusive.

Methods: we used sub-cellular fractionation and immunofluorescence staining to investigate the total expression and cellular distribution of Notch signaling components in MCF-7 cells treated with 17-β-estradiol, or estrogen-free medium.

Results: Our data shows that in cells treated with estrogen-free medium, Notch1 is located in the Lipid Rafts (LR) in the plasma membrane, along with Notch signaling components such as presenilin 1 (the catalytic subunit of γ-secretase), ADAM10 and Notch negative regulator NUMB. There is published evidence that γ-secretase is most active in lipid rafts.17-β-Estradiol modifies the membrane lipid composition and change the localization of Notch and Notch regulators (such as ADAM10, presenilin-1 and NUMB) at the plasma membrane, thereby altering the activation of Notch. These observations are consistent with a model in which the lipid composition of the plasma membrane is a critical factor in regulating the rate of Notch activation. FDA-approved cholesterol lowering drugs affect Notch signaling in ways consistent with this model.

Conclusions: Our findings indicate that endocrine therapy affects Notch signaling at least in part through alterations in membrane composition and lipid raft localization of Notch signaling components.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-22.
Abstract P6-04-23: Targeting of endocrine therapy resistance through combined mTOR and histone deacetylase inhibition
Authors: Ordentlich P , Flechsig S , Hoffmann J
Published: August 2014

Background: Primary and acquired resistance to endocrine therapy in advanced breast cancer presents a significant barrier to maximizing the clinical effectiveness of these agents. Preclinical data has demonstrated that up-regulation and/or constitutive activation of estrogen independent growth signaling pathways drive resistance to aromatase inhibitors and that targeting of these pathways is an effective strategy to overcoming the resistance. We recently reported in a randomized, placebo controlled phase 2 study that entinostat (ENT), an oral class 1 selective HDAC inhibitor, combined with exemestane extended PFS and OS in postmenopausal women with ER+ positive breast cancer that had progressed on a prior nonsteroidal aromatase inhibitor. Likewise, everolimus (EVE), a mTORi, combined with exemestane or tamoxifen (TAM) also significantly extended PFS in a similar patient population. In order to determine the potential benefit of combining ENT with EVE plus hormone therapy we have piloted a series of xenograft studies in primary human tumor models of TAM sensitivity and resistance.

Methods: Athymic nude mice bearing xenografts of tissue derived directly from patient tumors (MaCa4049 – TAM sensitive; MaCa3366/TAM – partially resistant to TAM) were treated with A) vehicle B) 2 mg/kg EVE C) 2 mg/kg EVE + 10 mg/kg TAM D) 15 mg/kg ENT E) 2mg/kg EVE + 10mg/kg TAM + 15 mg/kg ENT. Tumor samples taken at the end of the study were cryoconserved for analysis of gene and protein expression and protein phosphorylation.

Results: Growth of MaCa4049 tumors was inhibited in all treatment groups relative to vehicle in two independent studies conducted in this tumor model. Groups B, C and D demonstrated a similar level of activity while maximal inhibition was observed in Group E (combination of EVE + ENT + TAM). All treatments were generally well tolerated with weight loss similar in groups C and E. Treatment of MaCa3366/TAM tumors resulted in similar levels of tumor growth inhibition in groups B, C and E which were all highly effective in suppressing tumor growth in this model. As EVE alone was highly effective as a single agent in the MaCa3366/TAM model the contribution of ENT will be assessed through molecular analysis of tumors upon study completion. Efficacy results and analysis of tumor samples from completed studies will be presented to provide insight into mechanism of action for the triple combination.

Conclusions: Preclinical data demonstrating enhanced activity of entinostat combined with everolimus and hormone therapy provides the rationale for clinical investigation of this novel therapeutic approach to targeting hormone therapy resistance.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-23.
Abstract P6-04-24: Overexpression of protein kinase C alpha differentially activates transcription factors in T47D breast cancer cells in the presence of 17β-estradiol both in the 2D and 3D environments.
Authors: Pham TND , Asztalos S , Weiss MS , Shea LD , Tonetti DA
Published: August 2014

Background: Our lab has previously shown that overexpression of protein kinase C alpha (PKCα) results in a hormone independent, tamoxifen resistant phenotype in the T47D:A18 breast cancer cell line. Moreover, 17β-estradiol (E2) inhibits colony formation of T47D/PKCα in 3D Matrigel and tumor growth in vivo but not in the 2D environment (Zhang et al, Mol. Cancer Res, 2009). Differential transcriptional activation may account for the phenotypic changes observed in T47D/PKCα cells. In this study, we sought to identify transcription factors (TF) differentially activated in T47D:A18/neo versus T47D:A18/PKCα grown in 3D Matrigel in the presence and absence of E2, and compare these TFs with those activated in the 2D environment using a high-throughput screening (HTS) technology.

Methods: A system for rapid, non-invasive, large scale, dynamic quantification of TF activity was developed (Weiss et al, PLoS ONE, 2010). Reporter constructs were created containing a TF binding site that preceeds a basal promoter to produce firefly luciferase (Fluc) using a lentiviral backbone. The control construct contained only the basal promoter and was used for normalization. T47D/PKCα and T47D/neo cells were infected with lentiviral reporter constructs. Cells were seeded either on top of Matrigel or without Matrigel in 384 well plates in E2-free media. After 3 days, cells were treated with either E2 (10–9M) or vehicle (0.1% ethanol). Fluc activity was assessed by bioluminescence imaging at 24, 48 and 72 hours following treatment using Xenogen IVIS Spectrum imaging system.

Results: Initial findings showed differential transcriptional activities between the parental T47D/neo and T47D/PKCα cell lines at the basal level (without E2 treatment) in the 3D environment. Specifically, TFs involved in tumorigenesis (such as ETS1, STAT5) or the EMT process (such as SNAI1) were more highly activated in T47D/PKCα cells. Furthermore, E2 has a modulating effect on the activities of TFs in the two cell lines over time; observed both in the 2D and 3D environments. In particular, some TFs, such as Oct4, ETS1, and Stat5; were modulated (either induced or suppressed) by E2 only in T47D/PKCα cells.

Conclusion: These findings suggest that overexpression of PKCα alters TF activity in T47D breast cancer cells both basally and in response to E2. This HTS technology allowed us to reveal the dynamic regulation of TF activation in PKCα overexpressing T47D breast cancer cells. We are now beginning to understand the mechanism whereby PKCα may mediate differential response to E2 dependent on the microenvironment. This HTS approach is likely to lead to the identification of new therapeutic targets.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-24.
Abstract P6-04-27: EBAG9 immunoreactivity is a potential prognostic factor for poor outcome of breast cancer patients with adjuvant tamoxifen therapy
Authors: Shigekawa T , Ijichi N , Ikeda K , Miyazaki T , Horie-Inoue K , Shimizu C , Saji S , Aogi K , Tsuda H , Osaki A , Saeki T , Inoue S
Published: August 2014

Estrogen receptor-binding fragment associated gene 9 (EBAG9) is a primary estrogen responsive gene originally cloned from MCF-7 human breast carcinoma cells. Upregulation of EBAG9 expression has been observed in several malignant tumors such as advanced breast cancers, indicating that EBAG9 may contribute to tumor progression. In the present study, the immunohistochemical analyses of EBAG9 were performed using the specimens from breast cancer patients treated with tamoxifen as adjuvant therapy. To investigate the association between EBAG9 expression and the therapeutic effect of tamoxifen in breast cancer, we used adjuvant tamoxifen-treated breast cancer patient tissues from 3 institutions. The study was approved by the institutional review board. Patients with distant metastases within 5 years after surgery followed by tamoxifen treatment were defined as relapse cases, whereas those without distant metastases were defined as relapse-free cases. The immunoreactivity of EBAG9 was predominantly detected in the cytoplasm of breast cancer cells. Notably, EBAG9 immunoreactivity level was substantially elevated in the breast cancer samples from the relapse patients, and the positive immunoreactivity of EBAG9 was correlated with poor prognosis of the patients with adjuvant therapy. These results suggest that EBAG9 expression in tumor regions is associated with unfavorable prognosis of patients with adjuvant tamoxifen therapy and EBAG9 immunoreactivity can serve as a biomarker for adjuvant tamoxifen efficacy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-27.
Abstract P6-05-03: Targeted inhibition of recurrent PIK3CA mutations synergizes with bicalutamide in AR-expressing triple negative breast cancer
Authors: Lehmann BD , Bauer JA , Schafer JM , Tang L , Pendleton CS , Sanders ME , Pietenpol JA
Published: August 2014

Triple negative breast cancers (TNBC) are generally more aggressive than other types of breast cancer, with higher rates of relapse in the early stage and decreased overall survival in the metastatic setting. TNBC has been particularly difficult to treat given that the biology of the disease is not well understood, and until recently, molecular targets for therapeutic intervention have remained elusive. Lacking HER2 amplification as well as estrogen and progesterone receptors, TNBC appears to be a heterogeneous collection of biologically diverse cancers, which likely contributes to variable clinical outcomes for patients suffering from this disease. Using gene expression analyses, we recently identified six distinct molecular TNBC subtypes with unique biologies that include two basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem-like (MSL), and a luminal androgen receptor (LAR) subtype. We demonstrated that the growth of LAR cell line models was dependent on AR signaling, as siRNA-mediated AR knockdown or pharmacological inhibition by bicalutamide greatly diminished viability. In addition we previously demonstrated that LAR cell lines are highly sensitive to the PI3K/mTOR inhibitor NVP-BEZ235. Interestingly, we observed that all AR-positive TNBC cell lines examined contain PIK3CA mutations (H1047R) suggesting selection for deregulation of the PI3K pathways during evolution of AR expressing breast cancers. Further investigation of a panel of AR-positive TNBC tumors showed that PIK3CA kinase mutations are a frequent event in this TNBC subtype (69.2% vs. 26.9%) and not just an artifact of the LAR cell line models. We show that PI3K pathway activation plays a role in resistance to AR antagonists, as there are elevated levels of activated AKT in residual tumors after bicalutamide treatment of xenograft tumors. Furthermore, we demonstrate that genetic/pharmacological targeting of AR synergizes with PI3K/mTOR inhibition in both two- and three-dimensional cell culture models. Moreover, the combination of bicalutamide with PI3K/mTOR inhibitors GDC0980 or NVP-BEZ235 decreased tumor volume in mouse xenograft studies. Our preclinical data provide the rationale for pre-selecting AR-positive TNBC patients for future clinical trials evaluating the combination of AR antagonists with PI3K/mTOR inhibitors.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-03.
Abstract P6-05-04: Expression of the Androgen Receptor in triple negative tumors and its modulation by receptor tyrosine kinases and downstream pathways
Authors: Cuenca D , Montero JC , Morales JC , Prat A , Pandiella A , Ocana A
Published: August 2014

Background: Androgen receptor (AR) plays a central role in some tumor types like prostate cancer. Its expression and function in breast cancer is relatively unknown. Studies have linked tyrosine kinase (TK) receptors and their intracellular signaling pathways with AR status. In the present work we study the presence of AR in breast cancer human samples and how TKs can control its expression.

Methods: Tumor samples from patients were used to assess the expression of AR by western-blot. Membrane and intracellular kinases were evaluated using phosphoprotein arrays. Cell lines representative of all breast cancer subtypes were used to assess the expression of the AR and other signaling proteins using western-blot and phosphoprotein arrays. Proliferation studies with several kinase inhibitors were measured by MTT assays. We also interrogated publicly available gene expression microarray data sets with annotated clinical-pathological data.

Results: Across all the intrinsic subtypes of breast cancer, the expression of AR gene was found highly expressed in HER2-enriched and Luminal tumors and lowly expressed in Basal-like and/or triple-negative (TN) tumors. In TN tumors, PDGFRβ and EGFR were found highly activated, together with AKT and Erk1/2. A positive correlation was observed between activation of EGFR and PDGFRβ and the expression of AR. In vitro, AR protein expression varied across cell lines and was found more expressed in MCF7 (Luminal) and HS578T (TN), BT549 (TN), HCC70 (TN) and no expressed in HBL100 (TN) and MDA-MB231 (TN). Administration of the antiandrogen bicalutamide produced an anti-proliferative effect that was increased by association of an Erk1/2 inhibitor. Inhibition of the PI3K-mTOR pathway in BT549 and HS578T reduced the expression of AR but did not augment the action of bicalutamide. In TN tumors, AR gene and protein was found more expressed in older compared to younger patients.

Conclusions: In breast cancer, AR is expressed and is controlled by receptor TKs through the PI3K-mTOR pathway. An increased anti-proliferative effect is observed with the combination of bicalutamide and Erk1/2 inhibitors. AR expression correlates with age at diagnosis.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-04.
Abstract P6-05-06: Serum estradiol levels in postmenopausal ER+/PR- breast cancer are lower than those in postmenopausal ER+/PR+
Authors: Yamamoto Y , Ibusuki M , Goto H , Murakami K , Iwase H
Published: August 2014

(Background) Activation of growth factor receptor (GFR) signaling is related with ER+/PR- breast cancer (BC). Proportion of ER+/PR- phenotype in ER+ BC in postmenopausal patients is higher than that in premenopausal. However, this phenomenon cannot be explained by only GFR signaling activation. We hypothesized that ER+/PR- phenotype in postmenopausal patients has at least two subtypes, one is related with activation of GFR signaling and the other is related with suppression of PR expression due to low estrogenic stimulation.

(Purpose) The aim of this study is to clarify out hypothesis.

(Patients and methods) We measured serum E2 levels with radioimmuno assay (range of E2 levels: 1.3 to 50 pg/ml) in 154 postmenopausal ER+ BC patients. Cut-off levels of ER and PR were used 10% or more expression in tumor cells. We analyzed the correlation between serum levels of E2 and clinicopathological factors including PR expression. We also examined the relationship between HER2 expression levels and ki67 labeling index in ER+/PR- BC. In addition, we analysed the difference in ki67 LI between in premenopausal patients (n = 65) and in postmenopausal patients.

(Results) Median serum levels of E2 in ER+/PR+ phenotype (6.7 pg/ml) were significantly higher than in ER+/PR- phenotype (4.5 pg/ml)(p = 0.036). Serum levels of E2 was positively associated with body mass index (p = 0.01). However, there was no significant difference between serum E2 levels and other clinicopathological factors including tumor size, nodal status, nuclear grade, levels of ER expression and ki67 LI. In ER+/PR- phenotype, ki67 LI were varied (Median: 7.5%, range: 0.5 to 85%). Ki67 LI in Postmenopausal patients with ER+/PR- phenotype was significantly lower than that in premenopausal patients with ER+/PR- phenotype (p = 0.0022). Expression levels of HER2 was positively associated with ki67 LI(p = 0.002).

(Conclusions) ER+/PR- cancer in postmenopausal patients may have two types of phenotype. One is highly proliferative phenotype that is stimulated with GFR signaling. The other is low proliferative one that PR expression is suppressed by without sufficient estrogen supply. Serum levels of E2 might affect development of ER+ BC in postmenopausal women, especially in case of no activation of GFR signaling.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-06.
Abstract P6-05-08: Significant heterogeneity in ERalpha and PR receptor status in different distant breast cancer metastases of the same patient
Authors: Hoefnagel LDC , van der Groep P , Broers JE , van der Wall E , van Diest PJ
Published: August 2014

Purpose: Receptor conversion for estrogen receptor alpha (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in single distant breast cancer metastases has been described before. However, most patients develop multiple metastases. Heterogeneity in receptor status between different metastases of the same patient has not been studied, probably since such material is very rare. The consequence of such heterogeneity would mean that as many metastases as possible need to be biopsied to re-assess receptor status.

We therefore aimed to study heterogeneity of receptor status between different distant metastases of the same patient in a relatively large group by re-staining all primary tumors and metastases with current optimal immunohistochemical methods on full sections.

Material and methods: Formalin fixed paraffin embedded tissue of primary breast carcinomas and corresponding multiple (≥2) distant metastases from 38 female patients were collected from several pathology departments in The Netherlands and stained for ERα, PR and HER2 by IHC. We evaluated the heterogeneity of receptor status between the metastases and the correlation with the primary tumor.

Results: In the 4 cases that showed ER conversion, 2 (50%) showed heterogeneity in receptor expression between different metastases of the same patient. Of the 17 cases that showed PR conversion, 12 cases (71%) showed heterogeneity in expression between metastases. There were no changes in the receptor profile of primary breast cancer to their metastases for HER2 in this group.

Conclusion: In a significant number of metastatic breast cancer patients, there is heterogeneity in ER and PR receptor status between different breast cancer metastases of the same patient. This implies that as many metastases as possible need to be biopsied to re-assess receptor status and set the optimal indication for hormonal therapy in these patients. Alternatively, non-invasive assessment of the receptor status by molecular imaging may be an easier future way to assess receptor status of all metastatic lesions.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-08.
Abstract P6-05-12: Lack of Frequent Estrogen Receptor Mutation in Primary Breast Tumors
Authors: Oesterreich S , Kitchens C , Gavin P , Wu C-C , Riehle K , Coarfa C , Edwards D , Schiff R , Milosavljevic A , Lee A
Published: August 2014

The estrogen receptor alpha (ESR1) is a critical driver of breast tumorigenesis, and as such has been a target for therapy for many years. Early reports using Sanger sequencing and conformational assays reported that there were little if any mutations in ER-positive tumors, although a few somatic mutations have recently been described in tumors and cell lines. We set out to validate the authenticity of these reported somatic mutations by performing analysis of ER DNA sequence variants (DSVs) in 66 ER+ breast tumors, and 39 breast cancer cell lines. We utilized a combined approach of target capture sequencing of ESR1, and subsequent testing for novel and previously identified DSVs using an orthogonal mass spectrometry based sequencing approach. A DNA capture approach was designed to capture all exons, flanking splice sites, and the 3′ UTR of ESR1. DNA was captured and sequenced using SOLiD.

Using stringency with a cut-off with at least 2 variant reads, variants in at least 20% of the reads, and requiring evidence for the variants in both strands, we identified 4 previously reported SNPs (rs2077647; rs46432; rs1801132; rs2228480), and two previously reported somatic mutations H6Y and N532Y. We developed mass-spectrometry assays, including controls, for these DSVs, and in addition, we included 10 DSVs which had previously been reported as somatic mutations in breast (and other) cancer. Applying mass-spec based analysis to breast tumors and cell lines, we were able to confirm previously reported SNPs, and one previously reported mutations (H6Y). This mutation was found in one breast tumor and one cell line. None of the other reported somatic mutations could be confirmed in tumors or cell lines. Functional assays on the ESR1H6Y DSV failed to identify differences to wildtype receptor. The lack of a phenotype, and the infrequent occurrence of this DSV do not support a major driver role for ESR1H6Y.

This analysis suggests that ESR1 mutations are a rare event in ER+ primary breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-12.
Abstract P6-06-01: Lobular involution reduces breast cancer risk through downregulation of invasive and proliferative cellular processes
Authors: Radisky DC , Visscher DW , Stallings-Mann ML , Frost MH , Allers TM , Degnim AC , Hartmann LC
Published: August 2014

Background: Age-related lobular involution (LI) is a physiological process, usually accelerated with menopause, in which the epithelial tissue of the breast gradually regresses. Our previous analysis of a cohort of more than 14,000 women who had a benign breast biopsy at the Mayo Clinic revealed that women who had undergone LI were at significantly reduced risk of subsequent breast cancer development. In our studies of postmenopausal women specifically, we found that more than 40% had not completed the process of LI, and that the increased breast cancer incidence in older women was strongly associated with incomplete LI. We sought to determine the potential protective mechanistic effects of lobular involution.

Methods: We generated primary human mammary epithelial cell lines (HMECs) from breast biopsies of women who showed no LI (N = 6), partial LI (N = 7), or complete LI (N = 6). These cell lines were analyzed for proliferation in culture and profiled for genomic signatures using Affymetrix gene arrays. We also analyzed RNA isolated from a cohort of archived formalin-fixed, paraffin-embedded (FFPE) benign breast biopsy samples from postmenopausal women who had either completed the process of LI (N = 4) or who had not initiated LI (N = 8) for genomic signatures using Whole-Genome DASL Assays.

Results: We found that LI was associated with decreased cellular proliferation and inhibition of processes associated with tumor development and progression. Specifically, we found that HMECs derived from benign breast biopsies in which LI was not evident showed increased proliferation and elevated expression of Twist (p = 0.002), a transcription factor associated with epithelial-mesenchymal transition (EMT) and breast cancer development. We are assessing differentially expressed genes for association with breast cancer risk using a nested case:control group of benign breast biopsies from women in our cohort who subsequently developed breast cancer (N = 85) vs those who did not (N = 142) and for whom LI status at the time of initial biopsy was known.

Conclusions: LI may decrease cancer risk through inhibition of key tumorigenic processes. Identification of processes associated with the physiologic risk reduction seen with LI may provide insight into novel avenues for risk reduction.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-01.
Abstract P6-06-04: p53 and p16 as Modifiers of HERV-K in Invasive Pleomorphic Lobular Carcinoma and Classic Lobular Carcinoma of Breast
Authors: Dadfarnia Tahereh , Xu Ya , Hatch Sandra S , Dong Jianli , Eltorky Mahmoud A
Published: August 2014

Withdrawn by Author

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-04.
Abstract P6-06-05: Down-regulation of the circadian factor Period 2 by the oncogenic E3 ligase Mdm2: Relevance of circadian components for cell cycle progression
Authors: Liu J , Gotoh T , Vila-Caballer M , Santos CS , Yang J , Finkielstein CV
Published: August 2014

Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our body to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Circadian proteins also regulate genes involved in either cell division or death; and a perturbation of the balance among these processes leads to cancer development and progression.

A key aspect of cancer research is identifying new regulatory pathways involved in proliferation and differentiation of cell. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression, nor which, if any, of its components is either the ultimate effector that influences the fate of the cell.

Initial experiments were devoted to identifying new interacting partners for Per2 using a two-hybrid system. Interestingly, among the positive clones analyzed was the oncogenic protein Mdm2. This result was validated by immunoprecipitation of recombinant and endogenous Per2/Mdm2 complexes from unstressed cells. Pull-down assays using tagged-expressed proteins fragments and labeled proteins were later used to map the interacting regions between Per2 and Mdm2. Our results show Mdm2 binds to the central flexible region of Per2 known to interact with various protein partners. Thus, we hypothesized that binding of Mdm2 to Per2 might act by mediating its ubiquitination and therefore altering Per2 stability. We next examined the formation of the Mdm2/p53/Per2 complex by immunoprecipitation. Our data show anti-p53 antibody is able to co-immunoprecipitate Per2 and Mdm2. Moreover, in vitro and in vivo ubiquitination assays show that binding of Per2 to p53 prevented ubiquitination of p53 by Mdm2 without altering their binding. Immunofluorescence studies using H1299 cells (p53-) confirmed Per2 role in p53 stabilization and for localization. Overall our results suggest that Mdm2 modulates the stability of Per2 and p53 in unstressed cells, and might be responsible for the oscillatory levels of these proteins observed in a 24 h cycle.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-05.
Abstract P6-06-06: The therapeutic potential and hazard of exposure to Cerbera odollam leaf extracts
Authors: Chung F , Chan K , Lim YY , Li B
Published: August 2014

Background: Despite its significance in leading to the reduction of breast cancer mortality rates, therapeutic regiments for breast cancer still face two major problems: (1) the heterogeneity of the tumours and (2) the prevalence of drug resistance towards anti-estrogen therapy. The leaf extracts from Cerbera odollam were screened for active compounds with the potential to overcome these problems.

Methods: Hormone responsive (MCF7) and hormone-independent cell lines (MDA-MB-231) were treated with the fractionated methanolic extracts of C. odollam followed by analyses on cell cycle perturbations and gene expression changes resulting from the treatment. The stated analyses were performed by flow cytometry, semi-quantitative RT-PCR, western blotting and immunofluorescence. The active fraction was identified, upon which HPLC separation was performed to identify the putative active compound.

Results: Initial experiments revealed that a fractionated extract contains a potent cytostatic agent which is active towards hormone-responsive but not hormone-independent breast cell lines [the Estrogen Receptor-alpha (ERα) positive MCF7 cells remained static in growth for 10 days]. Comparison of the effects of the active and inactive fractions on MCF7 cells revealed key differences in cell growth and expression of ERα and its downstream regulated genes. The active compound was identified to be neriifolin (a cardiac glycoside) which also exhibited prolonged cytostatic effects (for 10 days upon treatment of 18.7 nM) similar to the active fraction. Gene expression analyses suggested that the ERα/Estrogen (E2) regulatory pathway and p53-dependent p21WAF1 transactivaton could be the cellular targets.

Conclusion: Although neriifolin could be a potential drug for breast cancer treatment, its severity in down regulation of the ERα/E2 regulatory pathway which is critical during female development indicates the potential health hazard from our exposure to C. odollam. We are now focusing on establishing assay to evaluate whether neriifolin could cause drug resistance (similar to tamoxifen) by silencing ERα expression through hypermethylation of the ERα promoter.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-06.
Abstract P6-07-01: Prognostic impact of the 21-gene Recurrence Score (RS) on loco-regional recurrence (LRR) of node-positive, ER-positive breast cancer patients (pts) treated with adjuvant chemotherapy: Results from NSABP B-28
Authors: Mamounas Eleftherios P , Tang Gong , Paik Soonmyung , Baehner Frederick L , Liu Qing , Jeong Jong-Hyeon , Kim Seong-Rim , Butler Steven M , Jamshidian Farid , Cherbavaz Diana B , Sing Amy P , Shak Steven , Julian Thomas B , Lembersky Barry C , Wickerham D Lawrence , Costantino Joseph P , Wolmark Norman
Published: August 2014

Withdrawn by Author

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-01.