bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract P5-07-05: Oncogene PELP1 regulates alternative splicing in breast cancer through PRMT6
Authors: Mann M , Vadlamudi RK 
Published: August 2014

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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 &lt;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

Abstract
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&lt;0.05) when grown in the absence of serum, greater invasive potential (p&lt;0.05) and formed larger and more rapidly growing colonies (p&lt;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&lt;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.
Abstract P5-09-13: HDAC inhibition re-sensitizes acquired hormone resistant cells to the cytotoxic effect of tamoxifen
Authors: Raha P , Thomas S , Thurn KT , Chen S , Munster PN 
Published: August 2014

Abstract
Breast cancer still continues to be a major cause of cancer related deaths in women, second only to lung cancer. Administering anti-estrogens and aromatase inhibitors, both in the adjuvant and metastatic settings, in estrogen receptor (ER) positive disease, is one of the most effective treatment strategies. However, prolonged exposure to these drugs leads to the emergence of resistance in about 40% of those who initially respond. Hence, the emphasis of a multitude of studies is to understand the underlying mechanisms of this acquired resistance and implementing means to overcome it.
Recent studies have implicated epigenetic modulation of gene expression in the development of resistance to the anti-estrogen tamoxifen. A clinical trial conducted by our group has demonstrated the efficacy of combining the HDAC inhibitor vorinostat with tamoxifen in patients who had progressed on prior anti-estrogen therapies and showed a total 40% clinical benefit (19% objective response and 21% stable disease for more than 6 months). In an effort to identify patients most likely to benefit from this novel therapy, we sought to elucidate the mechanism behind the clinical efficacy of this combination.
To this end, we have generated an in vitro tamoxifen resistant cell line (TAMR) by long-term exposure of MCF7 cells to 4-hydroxy tamoxifen. Significantly reduced anti-proliferative effect of tamoxifen and other anti-estrogens in TAMR compared to the sensitive MCF7 cells demonstrates the establishment of resistance to anti-estrogens in this cell line. In TAMR cells, addition of an HDAC inhibitor reverts resistance to tamoxifen. Although estrogen receptor (ER) expression in TAMR cells appears unaltered, the classical genomic signaling of ER in these resistant cells is suppressed and unresponsive to ligands, as deduced from transcription of ER response genes and luciferase assay of an ERE-luciferase construct. However, the ER remains important, since siRNA mediated depletion of ER inhibits cell growth in TAMR cells. Treatment with the dual EGFR/Her2 kinase inhibitor or an AKT inhibitor significantly inhibits the growth of these cells only when combined with tamoxifen, indicating the importance of crosstalk between pathways. Furthermore, Akt and mTOR protein and activities are down regulated by HDAC inhibition, which are further reduced when combined with tamoxifen. SiRNA mediated depletion of ER leads to reduced Akt and mTOR activities which suggests that ER may act as a driver, possibly through its non-genomic function at the plasma membrane activating members of the growth factor signaling pathways.
Studies are ongoing to further characterize the interaction of HDAC and ER inhibition on these signaling pathways and to determine their significance to resistance.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-13.
Abstract P5-09-14: Plant-derived estrogen receptorb agonists alone or in combination with aromatase inhibitors restore sensitivity in endocrine therapy resistant breast tumors
Authors: Samayoa C , Krishnegowda NK , Vadlamudi RK , Tekmal RR 
Published: August 2014

Abstract
Estrogen receptors (ER) play an important role in breast cancer. Over two-thirds of all breast cancers express ERa, and current endocrine therapies target its signaling. Antiestrogens (AE) block the binding of estrogen to ERα, while aromatase inhibitors (AI) inhibit local and systemic estrogen production. Both treatments improve outcomes for about 50% of patients with early or advanced ERα positive breast cancer. Unfortunately, therapeutic resistance frequently arises. There is a critical need to develop effective alternate strategies to prevent or delay the development of resistance to endocrine therapy and the resulting tumor progression. Both normal and tumor tissue express ERβ which has anti-proliferative activity and recent studies have identified several natural compounds that have the potential to function as ERβ agonists. Plant-derived ERβ-agonists, Liquiritigenin (Liq), from the plant Glycyrrhizae uralensis and S-equol, from the soy isoflavone daidzein, are currently in clinical trials for the treatment of vasomotor hot flashes associated with menopause. In this study we sought to determine if ERβ agonists alone or in combination with AI could resensitize Letrozole-resistant breast cancer cells in vivo. Xenografts were established using cells which model post-menopausal breast cancer. Endocrine therapy sensitive (MCF-7aro) and Letrozole resistant (LTLT-Ca) tumors were treated with either ERβ agonists or in combination with AI and progression was measured. In MCF-7aro tumors, ERβ agonist treatment reduced tumor volume and prolonged sensitivity to AI. In the therapy resistant tumors (LTLT-Ca), ERβ agonist treatment blocked tumor growth and restored sensitivity to AI therapy. To determine the molecular mechanism by which ERβ agonists inhibit tumor growth and prolong and restore sensitivity, qRT-PCR and western blot analysis was performed. Our results show changes in the levels of p53, NFkB, cyclin D1, and KLF5 in addition to other genes. To determine the role of ERβ agonists on the activation of these genes specific gene promoter-luciferase reporter constructs were used and the specificity of ERβ-mediated actions was confirmed using a siRNA approach. This study suggests that ERβ acts as a tumor suppressor by suppressing cell growth through the inhibition of cell cycle genes and by inducing apoptosis through the regulation of p53. In conclusion, this study demonstrates the potential role for ERβ agonists to improve adjuvant endocrine therapy to treat both hormone-responsive and hormone-resistant breast cancers.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-14.