bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P5-01-07: Select neutrophils inhibit breast cancer metastases in patients via chemokine-mediated mechanisms
Authors: Comen EA , Granot Z , Blum B , Coker C , Shah R , Seshan V , Norton L , Benezra R
Published: August 2014

Background: In both breast cancer patients and healthy women, we have previously demonstrated that select neutrophils found in breast cancer patients as opposed to healthy women are cytotoxic to breast cancer cell lines. (Granot Z et al. Cancer Cell. 2010) This work stemmed from our prior research in murine breast cancer models indicating that primary breast tumors can mobilize select neutrophils, termed Tumor Entrained Neutrophils (“TENS”); these entrained neutrophils have the unique capacity to inhibit metastatic seeding in the lung through cell-kill mechanisms. (Granot Z et al. Cancer Cell. 2010) In this study, we evaluated the relationship between select chemokines and neutrophil cytotoxicity in breast cancer patients versus healthy volunteers.

Methods: Neutrophils were purified from the blood of 75 randomly selected newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, and 47 healthy female volunteers with no history of cancer. Cytotoxicity was evaluated by incubating neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity was measured as a reflection of% cytotoxicity. Serum was also isolated from breast cancer patients and healthy volunteers. Based on prior experiments, we used the Millipore® Milliplex Human Cytokine Plex Kit to evaluate Il-1Ra, MCP-1 and TNFa in our serum samples in 50/75 of the cancer patients and 25/47 controls. We used multiple linear regression to develop a model to predict cytotoxicity as a function of the chemokines.

Results: In comparison to healthy volunteers whose mean neutrophil cytotoxicity to MDA-MB-231 cells was 6.5%, pre-operative breast cancer patients demonstrated a mean neutrophil cytotoxicity of 12.7%, p<0.0001. We then evaluated the serum chemokine levels of 50 of the breast cancer patients; 31/50 had high neutrophil cytotoxicity (>10%), and 19/50 had low neutrophil cytotoxicity (<10%). We compared the serum chemokine levels in the patients to the levels found in the 25 controls. Using a multiple linear regression model, we found that the levels of these three chemokines are associated with cytotoxicity (R2 = 0.126, p = 0.022). An ANOVA decomposition of the model suggested that Il.1RA was the most predictive (p = 0.018) followed by MCP.1 (p = 0.088) and TNF.alpha (p = 0.245).

Conclusion: Our work demonstrates the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with neutrophils from healthy women. We further demonstrate that select chemokines appear to be correlated with neutrophil cytotoxicity. We are currently evaluating the prognostic and therapeutic roles of cytotoxic neutrophils and their related chemokines in breast cancer patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-07.
Abstract P5-01-12: Prognostic implications of tumor-infiltrating lymphocytes (TIL) in association with PD-L1 expression, and serum cytokine levels in early breast cancer
Authors: Park IH , Kong S-Y , Ro JY , Kwon Y , Kang JH , Mo HJ , Jung S-Y , Lee S , Lee KS , Kang H-S , Lee E , Ro J
Published: August 2014

Background

Immune system has been known to influence the prognosis of breast cancer (BC). However, the relationship between immune modulating factor (PD-L1) and tumor infiltrating lymphocyte (TIL) profiles in breast cancer has yet to be revealed according to breast cancer subtypes. In addition, the effects of circulating cytokines on TILs have not been addressed.

Patients and methods

We investigated the relationship between the profiles of TILs and PD-L1 expression of the primary tumor tissue by immunohistochemistry with clinical outcomes in 253 patients who underwent surgery for early breast cancer at National Cancer Center from January 2001 to December 2005. Besides, the serum cytokines including IL-10, IL-18, IL-6, IFN-g, and TGF-β1 were measured at diagnosis. Clinical data including hormone receptors status, HER2 expression, disease free survival (DFS), and overall survival (OS) were collected.

Results

Median age of patients was 49 years (range, 32-74) and median follow-up was 8.5 years. One hundred eighty five (73.1%) patients had hormone receptor (HR) positive and 101 (39.9%) patients had node positive BC. CD8+ TILs were more abundant in low PD-L1 expressed tumor (P = 0.027), though there was no association between FOXP3+ TILs and PD-L1 expression (P = 0.585). A total number of TILs was higher in HR negative compared with HR positive BC (P = 0.061) and the expression of PD-L1 was more frequent in HR positive BC (P<0.001). In HR negative BC, there was a trend of longer DFS in patients with higher CD8+ TILs and low PD-L1 expression (P = 0.097). However, such association was not detected in HR positive BC patients. Among serum cytokines we examined, the higher levels of IL-18 were significantly associated with shorter DFS in HR negative BC (P = 0.006). In HR negative BC, higher CD8+ TILs with low PD-L1 expression and lower IL-18 were significantly related with better clinical outcomes when adjusted with other clinical factors (DFS, P = 0.032; OS, P = 0.048).

Conclusions

Lower PD-L1 expression in breast tumor was associated with higher CD8+ lymphocyte infiltration. Especially in HR negative BC, increasing CD8+ TILs with lower PD-L1 expression and lower serum IL-18 level were good prognostic factors. Further validation will be needed to establish the role of immune profiles in BC patients.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-12.
Abstract P5-01-13: Poly(I:C), an innate adjuvant receptor ligand, can induce the antitumor effects on human breast cancer cells
Authors: Inao T , Harashima N , Yamamoto Y , Harada M , Iwase H
Published: August 2014

[Background] Innate adjuvant receptors are expressed in immune cells and some types of cancers. If antitumor therapies targeting adjuvant receptors in cancer cells are established, it could be therapeutically useful because antitumor effects and activation of the immune system can be induced simultaneously.

[Objective]Poly(I:C) is one of innate adjuvant receptors ligands and triggers signaling through not only endosome Toll-like receptor (TLR) 3 but also cytoplasmic melanoma differentiation-associated gene (MDA) 5. In this study, we investigated antitumor effects of poly(I:C) on human breast cancer cell lines, and then elucidated the mechanisms.

[Methods] Three breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549) were used in this study. Poly(I:C) was transfected into these cancer cells, and cell viability and apoptosis were evaluated by WST8 assay and flow cytometry, respectively. The expression of adjuvant receptors and cell cycle-related molecules were examined by immunobotting. Autophagy was evaluated by immunoblotting and confocal imaging. In vivo antitumor effect was determined using a xenograft mouse model.

[Results] All three cell lines were positive for both TLR3 and MDA5. Poly(I:C) transfection drastically reduced their cell viabilities in a partially MDA5-dependent manner. Poly(I:C)-transfected cancer cells showed caspase-dependent apoptosis and growth arrest as a result of suppression of c-Myc and cyclinD1. Interestingly, poly(I:C) transfection was accompanied by autophagy, which is known to work cytoprotectively under starvation and stress, and knockdown of beclin-1, an essential molecule for autophagy, by RNA interference promoted poly(I:C) transfection-induced apoptosis. In vivo poly(I:C) transfection exerted the antitumor effect on xenografted MDA-MB-231 in nude mice.

[Conclusions] These results suggest that the innate adjuvant receptors are promising targets in human breast cancer and that autophagy plays a protective role in this treatment modality.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-13.
Abstract P5-03-02: Expression of RANK and RANK ligand (RANKL) in breast carcinoma and distinct breast epithelial cells from BRCA1 mutation carriers
Authors: Lindeman GJ , Visvader JE , Vaillant F , Mann GB , Soriano R , Branstetter D , Dougall WC
Published: August 2014

Background: Breast tumors in BRCA1 mutation carriers likely arise from luminal progenitor (LP) cells, previously shown to exhibit aberrant growth properties. Oophorectomy, and possibly tamoxifen, reduce breast cancer risk in BRCA1 carriers, potentially via inhibition of paracrine mediated signaling to stem/progenitor cells. RANKL is a major paracrine effector of progesterone's mitogenic action in mammary epithelium via its receptor RANK, and has a role in ovarian hormone-dependent activation of stem cells. Here we assessed RANK and RANKL expression in breast tumors and normal breast epithelial subtypes from women with mutations of BRCA1 (mBRCA1) or BRCA2 (mBRCA2).

Methods: RANK and RANKL expression in breast cancer or normal breast tissue samples with mBRCA1, mBRCA2 or wildtype (WT) BRCA1/2 were analyzed in formalin fixed paraffin embedded (FFPE) sections by IHC. kConFab and The Royal Melbourne Hospital Tissue Bank provided the samples used in this analysis; these samples were obtained with relevant IRB approval. RANK expression on normal breast epithelial cells was measured by flow cytometry. Antibodies against human RANK (N-1H8, N-2B10; Amgen) and RANKL (M366; Amgen) were used in both assays. Incidence of IHC staining was scored as a positive IHC signal of any intensity. The overall expression was generated using the H scoring method which is calculated as the staining intensity of the tumor (0-3) multiplied by the percentage of positively staining cells.

Results: Breast tumors from women with mBRCA1 had a higher incidence of RANK expression (68/162; 42%) compared with mBRCA2 (17/113; 15%) or WT (34/314; 11%) and higher overall H score (21.3) compared with mBRCA2 (8.0) or WT (3.4); RANKL expression did not vary greatly between groups: mBRCA1 (13/135; 10%), mBRCA2 (5/114; 4%), WT (23/212; 11%). In normal breast tissue, LP (Lin−EpCAM+CD49f+) and basal/stem cells (Lin−EpCAM−CD49fhi) expressed RANK on their surface. Similar expression patterns were seen in these epithelial subtypes from each BRCA1/2 genotype. Stromal cells (Lin−EpCAM−CD49f−) had minimal RANK expression.

Conclusions: RANK expression intensity and incidence scores are both enriched approximately 4-fold in breast tumors from BRCA1 carriers compared with other genotypes. Also, RANK is normally expressed in breast LP cells as well as the basal/stem cell containing population. Ongoing studies will assess functional regulation of LP or mammary stem cell activity by RANKL and determine if the RANKL/RANK signaling pathway affects the aberrant growth characteristics of these cells from BRCA1 mutation carriers.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-02.
Abstract P5-03-08: Mifepristone suppresses triple-negative breast cancer cell growth and cancer stem cell maintenance via down-regulating KLF5 expression
Authors: Liu R , Chen C
Published: August 2014

Triple-negative breast cancer (TNBC) is one of the most malignant breast cancers without effective therapeutic approaches currently. We previously reported that KLF5 is over-expressed in TNBC; KLF5 depletion significantly suppresses TNBC cell proliferation, survival and tumor growth. As a progesterone receptor antagonist, MIF has been reported to have anti-tumor activity in multiple hormone-dependent cancers, including luminal type breast cancers. Interestingly, we found that MIF is able to down-regulate KLF5 protein expression in TNBC cells, reduce cancer stem cell population and suppress cell growth of TNBC cells in vitro and in vivo. Similarly, we found KLF5 is also important for the maintenance of TNBC stem cell. We further demonstrated that MIF does not suppress KLF5 mRNA expression at all, but down-regulates KLF5 protein level via up-regulating miR-153 expression in TNBC cells. We found miR-153 mimics can down-regulate the KLF5 protein level in TNBC cells and decrease TNBC stem cell ratio similar to MIF does; consistently, miR-153 inhibitor rescued MIF-induced down-regulation of KLF5 protein expression and partially rescued the reduced cancer stem cell population by MIF treatment. Taken together, our findings suggest that MIF is able to inhibit TNBC growth and cancer stem cell population possibly via suppressing KLF5 expression; and our research may provide a new possible chemotherapeutic for the treatment of TNBC.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-08.
Abstract P5-03-12: Prognostic role of stem cell markers in triple-negative breast cancer (TNBC)
Authors: Song J , Apple S , Goodglick L , Mah VH , Kim SR , Alavi MT , Chia D , Deng X , Chang HR
Published: August 2014

Introduction: Triple-negative breast cancer (TNBC) is among the most aggressive types of breast cancer with frequent recurrence and death despite chemotherapy. Studies have shown that cancer stem cells play a key role in many of the drug resistant cancers including TNBC. In this study, we report the expressions of four stem cell markers in 158 TNBC tumors.

Design: Clinicopathological data were obtained from 158 triple negative breast cancers between 2002 and 2010. Tissue microarray was constructed and immunohistochemistry stains (IHC) for CD44 and CD24, CD 133 and ALDH1 were performed using the streptavidin-biotin method. Each set of TNBC consists of normal, ductal carcinoma in situ and invasive areas for assessment of IHC stains. Statistical analysis was done on the following parameters: Histological types, modified Bloom and Richardson grades, lymph-vascular invasion, lymph node status, tumor size, Ki67, presence of lymphocytic host response, age >50 or <50, BRCA status, prognosis (recurrence and death) and chemotherapeutic response. P values less than 0.05 were considered statistically significant.

Results: the mean age of this cohort of TNBC patients was 55 with 64% white, 13.2% black, 12.6% Asian/Pacific, 3.8% Hispanic/Latino and 1.9% other. The majority of these tumors (82.3%) were grade 3, 15.2% were grade 2, and less than 1% was grade 1. CD24, CD44 and CD133 expressions were significantly higher in TNBC than in normal breast tissues (p = 5.53 e-10, p = 1.14 e-11, p = 2.25 e-08 respectively). While both CD24 and CD133 expressions predicted poor survival (p = 0.073 and p = 0.043 respectively), neither was associated with recurrence. In contrast to CD24, CD44 and CD133 expressions, ALDH-1 expression was rare in TNBC and surrounding normal tissues. When expressed, ALDH-1 was associated with poor survival but cases were too few to reach significance. None of the four stem cell markers studied were associated with any of the conventional histopathologic features and BRCA1 or BRCA2 mutations.

Conclusions: Our study suggests that stem cell marker(s) may be prognostically important in TNBC patients. When it is fully characterized it may provide new insight in developing effective therapeutic strategies targeting stem cell related signaling pathways.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-12.
Abstract P5-03-13: ASPM as a link between mammary tissue architectures, Wnt signaling pathways, breast cancer stemness and aggressiveness
Authors: Tsai KK-C
Published: August 2014

Disruption of tissue microarchitectures represents an early step of tumorigenesis and the related molecular events may have important biological and clinical implications. Using a physiologically relevant model of tissue organization, we exploited the ubiquitous transcriptomic change associated with human glandular morphogenesis, thereby identifying a microcephaly-related gene ASPM that reciprocally links to mammary tissue architectures. We show that ASPM was functionally indispensible for the structural transformation of differentiated mammary acini and its depletion in breast carcinoma cells forced reversion of malignant tumor architectures. In vitro and in vivo functional assays, together with clinical correlative analysis, revealed that ASPM is an oncogenic factor that carries strong, independent and robust prognostic significance in breast cancer. Follow-on biochemical and functional studies demonstrated that ASPM serves as a scaffolding protein for upstream activators of beta-catenin, including notably dishevelled-2, whereby it regulates Wnt signaling pathways, cytoskeleton and cell locomotion. Interestingly, through its effect on the Wnt pathway activity, ASPM also critically regulate a subpopulation of breast cancer stem cells (CSCs) that exhibited high migratory potentials in vitro. Consistently, silencing of ASPM in breast CSCs attenuated the tumor growth and distant metastasis in mouse xenograft models of human breast cancer. Together, our results provide a novel mechanistic link between tissue architecture, Wnt signaling, breast cancer stemness and tumor aggressiveness (Supported in part by grants CA-101-PP-19 from National Health Research Institutes and DOH 102-TD-C-111-004 from Department of Health, Taiwan to K.K.T.).

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-13.
Abstract P5-03-14: Low dose ionising radiation effects in normal breast progenitor sub-populations
Authors: Kavanagh JN , Kissenpfennig A , Irwin GW , Prise KM
Published: August 2014

The human breast is a radiosensitive tissue and radiation exposure is proposed to contribute to mammary gland carcinogenesis through changes to stem and progenitor cells that result in aberrant proliferation, differentiation and genome instability. However, little is known about the low dose radiation response (LD IR) under conditions of relevance to environmental and occupational and medical imaging exposures.

This study utilises 2-D and 3-D breast models derived from two normal breast cell lines (MCF10A and HME1) and primary human mammary epithelial cells from reduction mammoplasty surgery, to investigate the mechanisms underpinning sensitivity of normal breast to LD IR and elucidate its role in breast cancer initiation and progression.

We have shown that DNA damage can be detected in both cell lines after doses <50 mGy and that an increased level of DNA damage foci at 1 h post LD IR is observed in a subpopulation of cells.

Using antibodies to cell surface markers suggested to be putative breast stem cell markers, we have performed Fluoresence Activated Cell Sorting (FACS) to isolate subpopulations from the MCF10A cell line. CD49fhi, EpCAM-, MUC1- cells thought to be progenitor like cells were found to be more resistant to LD IR than the EpCAM+, MUC1+ population.

As the pathways that govern survival/proliferation of stem and progenitor cells versus differentiation are interconnected, we have investigated if radiation resistance is also correlated with changes to breast cell differentiation. It was observed that LD IR modulated breast cell lineage markers in 2D cultures of both cell lines.

We subsequently found that radiation induces a decrease in MUC1 mRNA expression levels which coincides with increased Lef1 expression. Lef1 has been shown to promote Mre11 expression thereby conferring elevated DNA repair and radiation resistance in tumour cells.

Based on our findings, we hypothesize that progenitor cell populations within normal breast cell lines may be more efficient at detecting DNA DSBs resulting from LD IR leading to a pro-survival response. We are currently investigating these effects in primary mammary epithelial cell samples, using FACS to isolate progenitor subpopulations.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-14.
Abstract P5-04-11: CDK11p58 inhibits estrogen receptor a positive breast cancer invasion via integrinb3 through repressing ERa signaling
Authors: Chi Y , Wang L , Wang L , Wei P , Wu J
Published: August 2014

Background: The sex steroid estrogen plays a major role in the development and progression of breast cancer. Estrogen promotes breast cancer proliferation through a number of established pathways. CDK11p58, a Ser/Thr kinase which belongs to Cell Division Cycle 2-like 1 (CDC2L1) subfamily, is associated with cell cycle progression, tumorigenesis and apoptotic signaling. In the previous study, we found that CDK11p58 interacted with estrogen receptor a (ERa) in breast tumor cells and repressed ERa transcriptional activity through promoting its ubiquitin/proteasome-mediated degradation. Here we sought to investigate the critical role of CDK11p58 in the carcinogenisis of breast cancer.

Methods and results: We measured the expression of CDK11p58 in 132 pairs of breast cancer tissues and adjacent tissues by IHC and qPCR. The mRNA and protein level of CDK11p58 were significant higher in breast cancer tissues than those in normal breast tissues. Interestingly, low expression of CDK11 was associated with lymph nodes metastasis in patients (P<0.05). Using the ERa + cell lines ZR-75-30 and T47D, we found CDK11p58 could repress the migration and invasion of ERa positive breast cancer cells. We further demonstrated that mRNA of integrinb3 were dramatically repressed by CDK11p58 by the TaqMan® Metastasis Gene Expression Assays. The effect was CDK11p58 kinase activity dependent. Expression of integrinβ3 was highly related with ERa signaling. While overexpression ERa stimulates integrinβ3 expression, knockdown of ERa attenuates integrinβ3 expression. By the Dual-luciferase array and CHIP assay, we found estrogen response elements (ERE) indeed existed in integrinβ3 promoter. ERa bound to the ERE on the integrinβ3 promoter and increased its transcription.

Conclusions: Our findings provide mechanistic insights into how CDK11p58 regulated the expression of integrinβ3 to inhibit the cell invasion though repressing the signaling of ERa in a kinase activity dependent way. These data indicate that CDK11p58 is an antimetastasis gene product in breast cancer and regulation of integrinβ3 by CDK11p58 through ERa could be part of a signaling pathway that determines and explains breast cancer invasion and metastasis.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-11.
Abstract P5-05-04: Relationship between plasma estradiol levels and estrogen-regulated gene expressions in premenopausal estrogen receptor-positive breast cancers
Authors: Toyama T , Asano T , Sato S , Yoshimoto N , Endo Y , Hato Y , Takahashi S , Yamashita H
Published: August 2014

Background: Estrogen receptor (ER) and progesterone receptor (PgR) expression status in breast cancer are important factors in determining the use of adjuvant therapy. Plasma estradiol (E2) and progesterone vary markedly through the menstrual cycle. Recently, the levels of PgR expression were reported to be correlated with E2 at the time of surgery in Caucasian premenopausal breast cancer patients. Materials and methods: In this study, plasma E2 levels at surgery were examined for correlations with expressions of estrogen-regulated genes (ERGs), including PgR, and proliferation genes in 147 Japanese premenopausal ER-positive breast cancers. Plasma E2 and progesterone, FSH, and LH were measured on the day of surgery by ECLIA and mRNA expressions were analyzed for 4 ERGs (PgR, GREB1, TFF1, PDZK1), 2 progesterone-regulated (RANKL, cyclin D1) and 2 proliferation genes (MKI67, BIRC5) by RT-PCR using the TaqMan system. ER and PgR protein expression levels were estimated by immunohistochemistry. Results: Our results revealed that expressions of PgR mRNA and protein in breast cancer tissues were significantly correlated with plasma E2 levels at surgery (p = 0.005, p = 0.0165, respectively). Other ERGs were also significantly correlated with plasma E2 levels. However, there were no correlations between mRNA expressions of proliferation genes, MKI67 and BIRC5, and plasma E2 levels. Furthermore, mRNA expressions of the progesterone-regulated genes, RANKL and cyclin D1, were significantly correlated with PgR mRNA expression (p<0.0001, p<0.0001, respectively). Discussion: Significant correlations were seen in the expression of ERGs in Japanese ER-positive breast cancers according to the plasma E2 levels, which vary markedly through the menstrual cycle. Our results supported the previous report in Caucasian patients by other group. This variability may affect the interpretation of gene expression profiles incorporating ERGs such as the 21-gene recurrence score.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-04.
Abstract P5-05-05: Lower mitotic activity in BRCA1/2-associated primary breast cancers occurring after risk-reducing salpingo-oophorectomy
Authors: van Verschuer VMT , Heemskerk-Gerritsen BAM , van Deurzen CHM , Obdeijn IM , Tilanus-Linthorst MMA , Verhoef C , Schmidt MK , Koppert LB , Hooning MJ , Seynaeve C
Published: August 2014

Background Bilateral salpingo-oophorectomy reduces breast cancer (BC) risk by about 50% in BRCA1/2 mutation carriers when performed premenopausally. It has been hypothesized that growth activity of BCs originating after risk-reducing salpingo-oophorectomy (RRSO) is lower. We compared tumor characteristics and growth rates of BRCA1/2-associated primary BCs (PBCs) detected after RRSO with those of tumors originating without RRSO.

Methods From a cohort of 271 female BRCA1/2-associated patients with screen detected BC, 20 BRCA1/2 mutation carriers with PBC detected ≥12 months after RRSO were selected (RRSO group). Controls were 36 BRCA1/2 mutation carriers with PBC detected without RRSO (non-RRSO group) matched for age at PBC diagnosis (± 2.5 years) and for BRCA1 or BRCA2 mutation (intended matching ratio 1:2). Tumor growth rates, expressed as tumor volume doubling times (DT), were calculated. Pathology samples were revised for histological subtype, tumor differentiation grade, estrogen receptor (ER), progesterone receptor (PR) and HER2 status. Imaging examinations (magnetic resonance imaging (MRI), mammography) before and at BC diagnosis were revised.

Results Median age at PBC diagnosis was 52 years (range 35-67). MRI detected more BC than mammography in the RRSO-group as compared to the non-RRSO group (83% vs. 39%, P = 0.02). Tumor size at diagnosis was smaller in the RRSO group (11.0 mm vs. 17.0 mm, P = 0.01). Mitotic activity indexes (MAI) in the RRSO and non-RRSO group were 12 vs. 22 mitotic counts/2 mm2 (P = 0.02). No significant differences in differentiation grade, ER and HER2 status were found. PR-status was more often positive in the RRSO group without reaching statistical significance (38% vs. 13%, P = 0.07). Median DT in the RRSO group was 124 days (range 89-193) and 93 days (range 54-253) in the non-RRSO group (P = 0.47).

Conclusion BC occurring after RRSO in BRCA mutation carriers features a lower MAI, suggesting a less aggressive biological behavior. When confirmed in larger series, this may have consequences for BC screening protocols for BRCA1/2 mutation carriers after RRSO.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-05.
Abstract P5-05-08: Obesity suppresses estrogen receptor beta expression in breast cancer cells via a HER2-mediated pathway
Authors: Wiese MA , Bowers LW , Tekmal RR , Brenner AJ , Hursting SD , deGraffenried LA
Published: August 2014

Purpose: Increased levels of estrogen receptor beta (ERb) are correlated with an improved breast cancer prognosis. Our preliminary data suggests that obesity may decrease ERβ expression in certain breast cancer subtypes and may contribute to the more aggressive disease associated with obesity. This study aims to elucidate a potential mechanism(s) mediating this suppression.

Experimental Design: We utilized an in vitro model of obesity in which breast cancer cells were exposed to sera collected from postmenopausal breast cancer patients at the Cancer Therapy and Research Center at the University of Texas Health Science Center at San Antonio (UTHSCSA) and pooled by BMI category (obese (OB): ≥30 kg/m2; normal weight (NW): 18.5-24.9 kg/m2). Four human mammary tumor cell lines were used: SKBR3 (HER2 over-expressing, ERα negative), MCF-7 and ZR75 (ERα positive, HER2 negative) and MDA-MB-231 (triple negative), in addition to the mouse mammary tumor cell line, MMTV/neu (HER2 over-expressing). The effects of the sera on ERβ expression were assessed by qPCR. Protein expression levels were determined using Western blot analyses. Herceptin, a HER2 antagonist, was used to evaluate the role of HER2 in OB sera-induced ERβ modulation.

Results: OB sera exposure led to lower ERβ expression in SKBR3 and MMTV/neu cells in comparison to NW. Treatment of the SKBR3 cells with Herceptin at the time of OB sera exposure resulted in a three-fold increase in ERβ expression. In contrast, there was only a small increase in SKBR3 cell ERβ expression when Herceptin was added to NW sera, suggesting that the effects of the OB sera may be mediated by HER2 activity. In support of this hypothesis, exposure to OB versus NW sera did not modulate ERβ expression in MDA-MB-231, MCF-7 and ZR75 cells, which have very low levels of HER2. Lapatinib, another HER2 antagonist, will be used to inhibit HER2 signaling in MMTV/neu cells. On-going studies utilizing siRNA to the HER2 receptor aim to elucidate the HER2-regulated pathways contributing to the ERβ suppressing effects of OB sera in SKBR3 cells.

Conclusion: Based on our preliminary data, we hypothesize that obesity suppresses ERβ expression in breast cancer cells via a HER2-mediated pathway. Elucidation of the mechanism(s) mediating this effect could provide important insight into both the regulation of ERβ expression as well as how obesity promotes a more aggressive disease. Increased knowledge regarding the regulation of this effect may result in the identification of a new treatment target for the obese breast cancer patient population.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-08.
Abstract P5-07-05: Oncogene PELP1 regulates alternative splicing in breast cancer through PRMT6
Authors: Mann M , Vadlamudi RK
Published: August 2014

Background: Alternative splicing (AS) is essential in regulating gene expression and protein diversity; however, dysregulation leads to cancer development with tumor-specific splice variants that act by disrupting a tumor suppressor function. Protein arginine methylation is a modification involved in signal transduction, pre-RNA metabolism and transcriptional activation and deregulation of arginine methylase expression or functions can contribute to cancer progression via alternate splicing. PELP1 is a proto-oncogene, whose expression is associated with higher histological grade and shorter breast cancer specific survival, and PELP1 localization is a determinant of hormone sensitivity. This study focuses on examining the role of PELP1 in regulating gene expression and its possible role in alternative splicing.

Methods: ZR75 and MCF7 cells stably expressing PELP1-shRNA or overexpressing PELP1 were used in RNASeq and ChipSeq analysis. Gene differential expression lists were generated using DEseq and PELP1 regulated pathways were analyzed using Ingenuity Pathway Analysis (IPA). Cell proliferation, migration and ERE reporter gene assays were used to test the role of PRMT6 in PELP1's oncogenic functions. Immunoprecipitation, confocal microscopy, ChIP, and GST pull down assays were used to demonstrate PELP1-PRMT6 interactions. Exon specific RTqPCR assays and CD44 minigene reporter assays were used to demonstrate PELP1's role in splicing.

Results: RNA- and ChIP-sequencing results showed that the ERa coregulator PELP1 is a novel regulator of alternative splicing in breast cancer through the modulation of several genes involved in splicing including protein arginine methyltransferase 6 (PRMT6). PRMT6 plays a key role in coupling of transcription and alternative splicing by functioning as a transcriptional coactivator that can also regulate alternative splicing in a hormone independent manner. We discovered the novel complex of PELP1 and PRMT6 by co-immunoprecipitation. Biochemical assays revealed that PELP1 binds RNA, colocalizes with the splicing factor SC35 and promotes alternative splicing in reporter gene assays. PELP1 regulates the exon inclusion:skipping ratio of alternatively spliced exons in endogenous vascular endothelial growth factor (VEGF). PELP1's regulation of VEGF splicing is affected by the status of PRMT6. Interestingly, PRMT6 is needed for optimal oncogenic functions of PELP1. Further PELP1 has the potential to regulate the histone methyltransferase activity of PRMT6 and chromatin immunoprecipitation showed that PELP1 affects the enzyme recruitment and enrichment of the histone modification H3R2me2 at estrogen responsive genes.

Conclusions: Our findings show a role of PELP1-PRMT6 axis in ERα-mediated RNA splicing and their deregulation has implications for targeted therapeutics in ERα-driven breast cancer. This work was supported by the NIH grant CA095681 and NIH F31 Fellowship 1F31CA173909-01A1.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-05.
Abstract P5-07-08: Restoration of APC decreases tumorigenic potential in breast cancer cells
Authors: Cole JM , Prosperi JR
Published: August 2014

Mutations of the Adenomatous Polyposis Coli (APC) tumor suppressor gene have been reported in 5-70% of human breast cancers. APC is most well known for its role as a negative regulator of the Wnt/b-catenin pathway, where APC sequesters β-catenin and targets it for degradation via the proteasome. The breast cancer cell line, DU4475, has a single point mutation in the mutation cluster region of APC, coding for an early stop and subsequent truncated protein, lacking the β-catenin binding domain. Preliminary data using a TOPFlash reporter assay suggested that Wnt/β-catenin pathway was active, which was validated with immunofluorescence (IF) showing nuclear localization of β-catenin. Our overall hypothesis is that restoration of APC will decrease tumorigenic potential in DU4475 breast cancer cells. To test our hypothesis, we have generated DU4475 cells stably expressing GFP (control) (DU4475-GFP), a GFP-tagged internal fragment of APC that includes the β-catenin binding domain (DU4475-APCmid), and a GFP-tagged C-terminal fragment of APC that includes the microtubule binding domain of APC (DU4475-APCC-ter). After selection using G418, the GFP positive cells were sorted using a cell sorter. Results from TOPFlash reporter assay and IF demonstrated a modest decrease in Wnt/β-catenin activity in DU4475-APCmid cells. In addition, we observed decreased proliferation as measured in a growth assay in DU4475-APCmid cells compared to the control cells. We also observed a 17% increase in cleaved caspase-3 and only a minimal decrease in cell proliferation as measured by phospho-Histone H3 using IF in 3D cultures upon APCmid re-introduction. We also observed decreases in Cyclin D1, Cyclin B1, Early Growth Response 1, and HIF1α in DU4475-APCmid using real time PCR. These changes suggest that the re-introduction of the central domain of the APC tumor suppressor results in alterations in cell growth and gene expression changes indicative of an increased tumorigenic phenotype in vitro. Ongoing studies in the laboratory are dissecting the signaling pathways downstream of APC restoration to identify therapeutic targets that may be used to restore the functional alterations of mutant APC. These studies have generated a system to be utilized for in vivo tumor and metastasis formation under the control of APC mutation in a human cell culture model.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-08.
Abstract P5-08-09: Synergistic cytotoxicity of digoxin and 5-fluorouracil in doxorubicin-resistant breast cancer cell lines
Authors: Niu J , Zhang Y , Qiu Y , Clark S , Maddona MB , Chu F
Published: August 2014

BACKGROUND: Metastatic breast cancer is the leading cause of cancer death for women in the US. Cytotoxic chemotherapy remains one of the most important systemic treatment options for breast cancer patients. To enhance the tumor response to chemotherapy, attention has been focused on agents that reverse multidrug resistance (MDR) and increase the sensitivity of tumor cells to chemical drugs. Although many reversal drugs have been identified in vitro, their clinical application has been limited due to their own toxicity. It was reported that 5 years after mastectomy, the recurrence rate of breast cancer among patients on cardiac glycoside therapy, namely digoxin or digitoxin, was almost 10 times lower as compared with those who were not on cardiac glycoside therapy. Digoxin has also been shown to inhibit hypoxia-inducible factors (HIF) and block lung metastasis in a breast cancer model. Interestingly, digoxin was demonstrated to stimulate cell death in various cell lines including breast cancer. Therefore the aim of this study was to examine the reversal effect of combined digoxin and 5-fluorouracil (5-FU) on MDR resistance in human breast cell lines MCF-7 and MDA-MB-231 in vitro, as well as its mechanism of action.

METHODS: Wild-type and doxorubicin-resistant ER-positive (MCF-7 and MCF-7/DoxR) and triple negative (MDA-MB-231 and MDA-231/DoxR) breast cancer lines were used for this study. We have previously shown that both doxorubicin-resistant cell lines are also cross-resistant to 5-FU. Cell viability of both resistant cancer cell lines to 5-FU, digoxin, and both 5-FU with digoxin were compared to control at 96 hours of incubation both in normoxia and hypoxia using MTT assay. Western blot was used to quantify the level of HIF-1α and p-glycoprotein (P-gP).

RESULTS: In both doxorubicin-resistant cell lines, sub-IC50 concentration of digoxin together with 5-FU significantly decreased the cell viabilities at 96 hours compared to single agent digoxin and 5-FU. Under both normoxic and hypoxic conditions, the same synergistic cytotoxic effects were demonstrated. Western blot revealed that HIF-1α and P-gP were decreased in both doxorubicin-resistant breast cancer cells treated with the digoxin and 5-FU combination compared to single agent digoxin or 5-FU.

CONCLUSION: The combination of digoxin and 5-FU demonstrates a synergistic cytotoxic effect in doxorubicin-resistant breast cancer cell lines that is maintained under hypoxic conditions. This is, at least in part, via the inhibitory effects on both HIF-1α and P-gP. The combination of digoxin and 5-FU could be an effective clinical treatment strategy to overcome MDR in breast cancer. A phase II clinical trial is ongoing to test this hypothesis in patients with doxorubicin-resistant metastatic breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-09.
Abstract P5-09-04: The effect of nuclear factor kappa B activation on cell proliferation of estrogen receptor positive breast cancer cell lines with different HER2-status
Authors: Sas L , Dirix L , Vermeulen P , Van Dam P , Lardon F , Van Laere S
Published: August 2014

Introduction:Endocrine treatment is the mainstay of therapy for patients with Estrogen Receptor positive (ER+) breast cancer.Unfortunately, primary and secondary resistance to endocrine therapy are a major clinical problem. Previous studies have suggested a possible role for NFkB transcription factor family members in this resistance due to their effect on ER signaling. In this study, we aim to elucidate the interaction between both transcription factor families (ER and NFkB) and the consequence thereof on the growth pattern of ER+ cell lines.

Materials and methods:For this study, 2 ER+ breast cancer cell lines (i.e. MCF7 and MDA-MB-361) were selected based on a documented attenuated NFkB activity in these cell lines and a different ErbB2 status.Prior to treatment with estrogen (E2), TNFα (NFkB activation) and/or parthenolide (NFkB inhibition), cells were seeded in 48 well plates. Proliferation was measured by means of a crystal violet assay. ELISA experiments to measure NFkB DNA-binding in nuclear protein extracts and qRT-PCR for NFkB target genes was performed.

Results:Treatment of MCF7cells with different concentrations of TNFα initially increased cell proliferation, but after 48hrs, apoptosis occurred. Evaluation of NFkB DNA-binding showed a clear increase in RelA (p65), RelB (p62) and NFkB1 (p50) DNA-binding after 4hrs and up until but no longer than 24hrs of treatment. Results were confirmed by qRT-PCR for NFkB target genes.Due to the dynamics of NFkB activation secondary to TNFα treatment, we limited further experiments to 24hrs of treatment. Combination of E2and TNFα gave an additive effect in MCF7 cell proliferation compared to E2 alone, an effect counteracted by parthenolide in a concentration dependent manner. In the ErbB2+ cell line MDA-MB-361 our initial results suggest that addition of TNFα does not affect E2-driven cell proliferation.

Conclusion: Our results so far suggest a different, ErbB2-dependent role for NFkB on the proliferation of ER+ breast cancer cells. Interestingly, the effect of NFkB seems to depend on both RelA- and RelB-dependent pathways. Experiments are currently being performed to elaborate on the present results.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-04.
Abstract P5-09-09: ESRP1 and ESRP2 expression in tamoxifen resistance
Authors: Gökmen-Polar Y , Goswami CP , Gu X , Nakshatri H , Badve S
Published: August 2014

Background: Current standard of care therapies with antiestrogens targeting estrogen receptor α (ER) signaling improve disease-free survival (DFS) in early-stage of breast cancer. However a significant number of cases exhibit de novo or acquired endocrine resistance and recur. It is thus important to identify novel targets of resistance and select these patients for additional therapeutic options which might include continued/extended hormonal therapy. The Oncotype Dx recurrence score (RS) in current practice predicts the likelihood of distant recurrence in tamoxifen-treated patients with node-negative, ER+ breast cancer. However, Oncotype Dx does not provide the mechanistic basis for endocrine resistance. In this study, we aimed to investigate the impact of two epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) expression on recurrence and endocrine resistance such as tamoxifen of ER+ breast cancer.

Methods: Publicly available gene expression datasets were analyzed for the overexpression of ESRP1 and ESRP2 in breast cancer. To validate the in silico findings for the ESRP1 and ESRP2 expression, we further performed quantitative real-time RT-PCR (qRT-PCR) in a cohort of 60 paraffin-embedded ER-positive node-negative breast carcinomas with low, intermediate, and high (19, 21, and 20 cases, respectively) Oncotype DX scores. To further determine the correlation between endocrine resistance and ESRP1/ESRP2 expression, we evaluated their expression levels in an established in vitro model of ER+/tamoxifen-resistance (MCF7/LCC2: an acquired tamoxifen resistant model of human breast cancer).

Results: Using publicly available breast cancer gene expression datasets, we have identified that overexpression of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) correlates with worse prognosis in ER+ significantly, but not in ER- breast cancers. qRT-PCR analysis further revealed that ESRP1 and ESRP2 expressions are positively correlated with high Oncotype Dx scores (P <0.05). In vitro, ESRP1 and ESRP2 levels were increased 7.48-fold (P = 0.008) and 2.98-fold (P = 0.0007) in ERα-positive cells with acquired tamoxifen resistance (MCF-7 LCC2), respectively. Thus, ESRP1 expression was slightly elevated more than ESRP2. Furthermore, both ESRP1 and ESRP2 levels did not change in response to E2 alone or E2 and tamoxifen treatment in combination.

Conclusion: These findings suggest a role for the elevated expression of ESRP1 and ESRP2 in tamoxifen resistance and recurrence of ER+ breast cancer. Further studies are ongoing to determine their mechanistic role in ER+ cancer and methods of targeting ESRP1 & 2.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-09.
Abstract P5-09-11: BP1, a homeoprotein, regulates estrogen receptor alpha and induces estrogen independence
Authors: Berg P , Kirolikar S , Ginsburg E , Schwartz A , Simmens S
Published: August 2014

Introduction: BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors. Moreover, 100% of inflammatory breast tumors are BP1 positive. These data led us to define the mechanism of BP1-related tumorigenesis and aggressiveness in breast cancer.

Materials and Methods: MCF-7/O1, O2 and O4 cells overexpressing BP1 and control V1 and V2 cells were tested for growth in estrogen free media, malignant potential and invasiveness using cell viability assays, soft agar assays and matrigel assays, respectively. To determine the influence of BP1 overexpression on tumor characteristics, empty vector cells (V1) and overexpressor cells (O2 and O4) were injected into the fat pads of athymic nude mice. Mice were supplemented with estrogen pellets or were unsupplemented. Chromatin immunoprecipitation assays (ChIP) were used to validate the binding of pBP1 to ERa and EP300. The effects of BP1 expression on ERα and EP300 were investigated using immunoblotting and qRT-PCR. Effects of BP1 overexpression on tamoxifen sensitivity were measured using the MTT assay.

Results: Cells overexpressing BP1 showed higher viability (p<0.05) when grown in the absence of serum, greater invasive potential (p<0.05) and formed larger and more rapidly growing colonies (p<0.0001) compared with cells containing the empty vector. Tumors were larger in mice receiving O1, O2 or O4 cells than in mice receiving V1 or V2 cells (p<0.0001). There was also a positive correlation between BP1 mRNA levels and tumor size in patients (p = 0.01). 20% of mice injected with O2 or O4 developed tumors in the absence of estrogen, in contrast to control mice which did not develop tumors. Several mechanisms of estrogen independence related to BP1 were established: (1) an indirect mechanism, in which BP1 stabilizes ERα protein by transcriptional activation of p300, and (2) a direct mechanism, in which BP1 binds to the first intron of ERα and upregulates ERα RNA and protein expression. In addition, breast cancer anti-estrogen resistance 1 (BCAR1) RNA expression was higher in O2 as compared to V1 cells. Consistent with these findings, O2 cells exhibited increased proliferation when treated with tamoxifen, while V1 cells showed growth inhibition.

Conclusion: High BP1 levels can lead to estrogen independence in ER positive breast cancer cells and tumors in mice by at least two mechanisms, indirect and direct, and are associated with resistance to tamoxifen. These results suggest that BP1 may be an important therapeutic target in ER positive breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-11.