The purpose of this study was to investigate the differential expression and function of organic anion-transporting polypeptides (OATPs) in breast epithelial and breast cancer cells. Estrone-3-sulfate (E3S), a substrate for 7 of 11 OATPs, is a predominant source of tumor estrogen in postmenopausal, hormonedependent patients with breast cancer. Overexpression of certain OATPs (e.g., OATP1A2) reported in breast tumor tissues compared with surrounding normal tissues could contribute toward two to three times higher tumoral E3S concentration. Little is known about expression and function of other OATP family members among breast epithelial and breast cancer cells. We therefore compared gene and protein expression of seven OATPs (OATP1A2,OATP1B1,OATP1B3,OATP1C1,OATP2B1,OATP3A1, and OATP4A1) in immortalized breast epithelial cells (MCF10A), hormone-dependent breast cancer cells (MCF7), and hormoneindependent breast cancer cells (MDA/LCC6-435, MDA-MB-231, and MDA-MB-468) by quantitative polymerase chain reaction and immunoblotting, respectively. Expression of solute carrier superfamily encoding for OATPs (SLCO) 1A2, 1B1, 1B3, 2B1, and 3A1 is exclusive, similar, or significantly higher in cancer cells compared with MCF10A cells. Protein expression of OATPs is found to be either exclusive or higher in cancer cells compared with MCF10A cells. Specificity of OATP-mediated E3S uptake is observed only in cancer cells, with the highest total uptake in MCF7 cells. Transport kinetics of E3S uptake demonstrates transport efficiency that is 10 times greater in the MCF7 cells than in the hormone-independent cells. These data suggest that OATPs could be a novel therapeutic target for hormone-dependent breast cancers, particularly in postmenopausal patients, where the major source of tumor estrogen is E3S.

The current study investigates the potential of estrone-3-sulphate (E3S) as a ligand for targeting Organic Anion Transporting Polypeptides (OATP), a family of membrane associated uptake transporters, for detection and diagnosis of hormone dependent breast cancers. E3S, an OATP substrate, is a predominant source of tumour estradiol in post-menopausal patients. To assess the potential of E3S as a ligand, distribution of exogenous E3S was determined at the whole body, tumour and cellular levels in murine models of hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancers. The highest levels of tumour uptake were observed at 6 h post injection (p.i) with significant difference (p = 0.04) between the level in MCF-7 (13.9±3.1%ID/g) and MDA-MB-231 (10.4±1.1%ID/g) (%ID/g: percentage of the total injected dose per gram tissue). The highest tumour-to-blood ratios (MCF-7∶7.4±1.2; MDA-MB-231∶9.1±2.1) were observed at 48 p.i., and highest tumour-to-muscle ratios (MCF-7∶10.7±1.5; MDA-MB-231∶3.8±0.7) were observed at 6 h p.i. Analogous to total tumour uptake, ex vivo tumour cell uptake at 2 h p.i. was 6 fold higher in MCF-7 in comparison to MDA-MB-231 tumour cells. Blocking studies, conducted by pre-administration of 100-fold excess E3S, resulted in significantly lower (MCF-7: p = 0.01; MDA-MB-231: p = 0.02) tumour uptake in both xenograft models, suggesting the involvement of an active carrier-mediated process. The expression of OATP1A2 was detected in tumour sections from both xenografts, with significantly higher expression (p = 0.002) in the MCF-7 xenografts. Overall, the higher tumour uptake and tumour-to-muscle ratio, alongside the higher expression of OATP1A2, in the MCF-7 xenograft model suggests the potential of E3S to serve as a novel ligand for targeting hormone dependent breast cancers.

Two-thirds of newly diagnosed hormone-dependent (HR?) breast cancers are detected in post-menopausal patients where estrone-3-sulphate (E3S) is the predominant source for tumour estradiol. Understanding intra-tumoral fate of E3S would facilitate in the identification of novel molecular targets for HR? post-menopausal breast cancer patients. Hence this study investigates the clinical expression of (i) organic anion-transporting polypeptides (OATPs), (ii) multidrug resistance protein (MRP-1), breast cancer resistance proteins (BCRP), and (iii) sulphatase (STS), 17β-hydroxysteroid dehydrogenase (17β-HSD-1), involved in E3S uptake, efflux and metabolism, respectively. Fluorescent and brightfield images of stained tumour sections (n = 40) were acquired at 4× and 20× magnification, respectively. Marker densities were measured as the total area of positive signal divided by the surface area of the tumour section analysed and was reported as % area (ImageJ software). Tumour, stroma and non-tumour tissue areas were also quantified (Inform software), and the ratio of optical intensity per histologic area was reported as % area/tumour, % area/stroma and % area/non-tumour. Functional role of OATPs and STS was further investigated in HR? (MCF-7, T47-D, ZR-75) and HR-(MDA-MB-231) cells by transport studies conducted in the presence or absence of specific inhibitors. Amongst all the transporters and enzymes, OATPs and STS have significantly (p < 0.0001) higher expression in HR? tumour sections with highest target signals obtained from the tumour regions of the tissues. Specific OATP-mediated E3S uptake and STS-mediated metabolism were also observed in all HR? breast cancer cells. These observations suggest the potential of OATPs as novel molecular targets for HR? breast cancers.

Introduction-Organic Anion Transporting Polypeptides (OATP) are a family of membrane associated transporters that facilitate estrone-3-sulphate (E3S) uptake by hormone dependent, post-menopausal breast cancers. We have established E3S as a potential ligand for targeting hormone dependent breast cancer cells, and in this study sought to prepare and investigate radioiodinated E3S as a tool to study the OATP system. Methods-2-and 4-Iodoestrone-3-sulfates were prepared from estrone via aromatic iodination followed by a rapid and high yielding sulfation procedure. The resulting isomers were separated by preparative HPLC and verified by 1 H NMR and analytical HPLC. Transport studies of 2-and 4-[ 125 I]-E3S were conducted in hormone dependent (i.e. MCF-7) and hormone independent (i.e. MDA-MB-231) breast cancer cells in the presence or absence of the specific transport inhibitor, bromosulfophthalein (BSP). Cellular localization of OATP1A2, OATP2B1, OATP3A1 and OATP4A1 were determined by immunofluorescence analysis using anti-Na + /K + ATPase-α (1:100 dilution) and DAPI as plasma membrane and nuclear markers, respectively. Results-Significantly

Background: Recent discoveries of recurrent and targetable gene fusions in breast cancer suggest the need to characterize the functional significance of such genomic aberrations within larger cohorts. We quantify fusion transcript expression in patient samples using RNASeq and evaluate their functional significance using biological pathway enrichment analysis. Methods: We sequenced transcriptomes of core biopsy RNA from 97 breast tumors obtained from brief-exposure preoperative clinical trials BrUOG 211A/211B. HER2- patients were treated with brief exposure to bevacizumab (B) or nab-paclitaxel (nP) followed by treatment with B/nP/carboplatin while HER2+ patients received brief exposure to trastuzumab (T) or nP followed by T/nP/carboplatin. Paired-end sequencing on 55 baseline biopsies and 42 post-exposure biopsies using amplified total RNA yielded 55 million reads on average per sample. We assigned RNASeq-based PAM50 subtypes for each of the samples using standard methodology. Fusion transcript abundance was evaluated using two independent pipelines, TopHat and deFuse, due to their complementary strategies in fusion detection. We eliminated fusions of genes with their respective pseudogenes as likely false positives arising due to alignment artifacts. TopHat fusion calls with total supporting reads ≥10 and deFuse calls with probability of fusion ≥0.7 were considered reliable. Results: We identified high confidence gene fusions, detected by both TopHat and deFuse, in 30 of the 55 baseline biopsies (54.4%), with 3.3 fusions on average per sample and a maximum of 10. Fusions were predominantly associated with chromosomal aberrations (75%), with putative deletions responsible for 32% of fusions and translocations responsible for 43%. We find a high level of fusion transcript heterogeneity within breast cancers, detecting a total of 80 fusions across the 30 samples with only three fusions recurrent in two samples with high expression in each: MDN1-GAS5 in two basal breast cancers, KRAS-GRIP1 and ITPR2-CCDC91 in two LumB cancers. Several cancer-related genes were found to be fusion partners: AKT3-SMYD3, CREB1-PPP1R1C, FLOT2-TOP2A and FOXC1-ARID1B. Pathway analysis of the fusion genes at baseline revealed enrichment of proteasome (p = 0.000752), tight junction (p = 0.027), insulin signaling (p = 0.0284) and melanogenesis (p = 0.05) pathways after multiple testing correction (FDR≤0.25). We looked for modulation of gene fusions upon brief exposure to therapy in 18 patients and found a majority of the baseline fusion transcripts to be present post-brief exposure in 44% of the patients, irrespective of therapy regimen. Conclusions: We find that gene fusions in breast cancer are highly heterogeneous but are enriched with cancer-related pathway genes. This is the first study to report a novel gene-lincRNA fusion transcript (MDN1-GAS5). We are currently validating the fusion calls using qRT-PCR. The heterogeneity of detected fusions suggests that multiple mechanisms could underlie the selective advantage of tumor cells expressing fusion transcripts. The brief-exposure preoperative paradigm provides a unique opportunity to evaluate modulation of fusion transcripts that can shed light on their functional importance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-07.