L-Z Sun

Abstract P5-03-03: Effect of rapamycin on the function and transformation of aged murine mammary stem cells

Age is the number one risk fact for breast cancer with only 5% of all breast cancers occurring in women under 40 years old and age specific incidence of invasive breast cancer increases from less than 1.5% at age 40, to about 3% at age 50 and over 4% by age 70 in American women. Recent research implicated that adult mammary stem cells (MaSCs) might be responsible for the initiation and progression of certain types of breast cancer. Our preliminary study showed that aging is associated with a significant increase of MaSC frequency, but with a functional decline of self-renewal and differentiation as well as increased neoplastic transformation potential. These findings indicate that aged MaSCs might be the precursors of preneoplastic lesions and serve as the cell of origin for malignant transformation in breast tissue. Therefore, intervention of MaSC aging process could be an effective method for cancer prevention. The drug of rapamycin has been shown to extend life span and ameliorate age-related pathologies (e.g., cancer) in murine models, and a recent study suggested that rapamycin9s anti-aging effect may partially act through enhancing the function of tissue-specific stem cells. It is unknown whether rapamycin treatment will also enhance the function of aged MaSCs and decrease their transformation potential. In this study, we fed C57BL/6 mice with microencapsulated rapamycin-containing food (14 mg/kg, food designed to deliver ∼2.24 mg of rapamycin per kg body weight/day to achieve about 4 ng/ml of rapamycin per kg body weight/day) or control diet with empty capsules for 2 year (starting at 2-month old) or 5-10 days (starting at 25.5-month old) and then isolated primary mammary cells at 26-month old for MaSC quantification using an in vitro mammosphere formation and 3D-ECM sphere differentiation assay as well as by the in vivo cleared mammary fat pad transplantation assay. Our findings indicate that short-term (5-10 days) or long-term (> 2 year) rapamycin treatment reversed phenotypic changes associated with aged MaSC, which was mainly characterized by decreased luminal-to-basal cell ratio and increased MaSC frequency. Histological analysis of regenerated glands of aged MaSCs derived from control and rapamycin-treated mice showed a significant decrease of early neoplastic transformation potential in rapamycin-treated group. Subsequent in vivo serial transplantation and mating experiments revealed that rapamycin treatment reverted aged MaSCs more resemble to young MaSCs in self-renewal/differentiation function during regeneration and improved lobulo-alveolar differentiation function for lactation. In conclusion, our findings suggest that rapamycin can rejuvenate the function of aged MaSCs as well as reduce their incidence of preneoplastic transformation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-03.

Abstract P5-04-04: Significance of PELP1/HDAC2/microRNA-200 regulatory network in EMT and metastasis of breast cancer

Tumor metastasis remains a significant clinical problem and is the leading cause of death among breast cancer patients. Estrogen receptor (ER)-coregulators play an essential role in cancer progression and metastatic tumors express increased levels of coregulators. Proline glutamic acid rich protein (PELP1) is an ER coregulator, its expression is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. MicroRNA (miR) mediated regulation of tumorigenesis is emerging as a new paradigm in cancer biology and widespread misexpression of miRs has been reported in breast cancer. The objective of this study is to examine the mechanism and therapeutic significance of PELP1 regulation of miRs leading to breast cancer metastasis. We have used both ER+ve (ZR75, MCF7) and ER-ve (MDAMB231, MDAMB468) models that either stably overexpress PELP1 or PELP1shRNA. Boyden chamber, and invasion assays demonstrated that PELP1 down regulation significantly affect migration of both ER+ve and ER-ve cells. Epithelial to Mesenchymal Transition (EMT) real time qPCR Array studies identified PELP1 modulate expression of EMT genes Snail, Twist, ZEB1, ZEB2, Vimentin and MMPs. Importantly, whole genome microRNA array analysis using PELP1 model cells revealed that miR200a and miR141 were significantly upregulated in cells expressing PELP1-shRNA compared to control cells. Accordingly, over expression of PELP1 in low metastatic model cells decreased expression of miR200a and miR141. PELP1 regulation of miRs was further confirmed by ZEB1 and ZEB2 3′ UTR luciferase reporter assays. ChIP analysis revealed recruitment of PELP1 to the proximal promoter region of miR-200a and miR141 and promoter reporter assays further confirmed PELP1 regulation of miRs. Interestingly, PELP1 down regulated expression of miR200a and miR141 by promoting repressive chromatin modifications via HDAC2. Supporting this, HDAC inhibitors reversed PELP1 driven repressive effects. Further, ectopic expression of miR200a and miR141 mimetic decreased PELP1 mediated invasion/metastatic functions. Prognostic significance of PELP1-miRNA axis was determined using Tissue micro-array (TMA) and in situ hybridization (ISH assays) of Locked Nucleic Acid (LNA™)-based microarray approach in 102 human breast tumors. To test therapeutic potential in vivo , we have generated ZR-PELP1- and MCF7-PELP1-shMIMIC of miR200a and miR141 stable cells. In vitro gene expression and Boyden chamber assays using these model cells revealed that shMIMIC of miR200a and miR141 reversed PELP1 mediated alterations in gene expression and reduced PELP1 driven migration/invasion. Proof of principle studies using IVIS imaging of nude mice based assays of GFP-Luc labeled cells demonstrated therapeutic efficacy of miRIDIAN shMIMIC of miR200a and miR141 on PELP1 driven in vivo metastasis. Collectively, these novel findings demonstrate for the first time a previously unknown role for PELP1 in epigenetically controlling the functions of tumor metastasis suppressor miR-200a and miR141. These results suggest that PELP1-miR axis may be crucial stimulus for promoting EMT and breast cancer metastasis. This study is funded by NIH T32CA148724 Postdoc Fellowship Grant. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-04.

Abstract P5-03-04: Effect of aging on the function and transformation of murine mammary stem cells

Epidemiological studies have shown that the risk of getting breast cancer progressively increases with age. A woman is 100 times more susceptible to develop breast cancer in her 60s than in her 20s. A better understanding of altered cellular and molecular mechanisms leading to the development of sporadic breast cancers as a result of aging are urgently needed for its prevention and treatment in growing population of older women. Recent research implicated that adult mammary stem cells (MaSCs) might be responsible for the initiation and progression of certain types of breast cancer. But no studies have been reported on how lifelong exposure of MaSCs to endogenous and environmental stresses during aging compromises their self-renewal and differentiation function and predispose them to neoplastic transformation either spontaneously or after carcinogen exposure. In our study, we have investigated the effect of progressive aging and carcinogen exposure on MaSC number and function in a murine model. The FACS isolated MaSC enriched basal cells, characterized by their immunophenotype (Lin − CD49f high CD24 med ) were utilized to evaluate MaSC frequency and function during aging by in vitro mammosphere formation and 3D-ECM sphere differentiation assay as well as by in vivo cleared mammary fat pad transplantation (IVT). The results of our study showed that the percentage of MaSCs analyzed by both FACS profile and in vitro assays, increased steadily with increasing age observed at 2, 7, 17 and 25 month-old C57BL/6 mice. Subsequent IVT using mammosphers or 3D structures formed by young (4 months) and old MaSCs (28 months) derived from C57BL/6 mice showed similar in vivo functional mammary gland regenerative capacity indicating similar self-renewal/differentiation ability between young and old MaSCs. However, we found that the regenerated glands from old MaSCs had significantly higher number of spontaneous pre-neoplastic lesions (52.0%) than those from young MaSCs (18.3%). Significantly, MaSC frequency was also increased by the in vivo exposure of a carcinogen, N-Methyl-N-Nitrosourea (MNU, 25 mg/kg body weight, once a week for three consecutive weeks), in old mice with no effect on total mammary cell number. Furthermore, the old MaSCs expanded by MNU treatment were still capable of in vivo regeneration with similar success rate as that of normal old MaSCs. But, we observed increased frequency of hyperplasia and dysplasia in the regenerated glands initiated by MNU treated old MaSC. Our results indicate that MaSCs might be the precursors of preneoplastic lesions and old MaSCs appear to be predisposed to premalignant transformation, which is enhanced by the exposure to a DNA damaging agent. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-04.

P2-03-05: Attenuation of TGF-beta Signaling Suppresses Premature Senescence in a p21-Dependent Manner and Promotes Oncogenic ras-Mediated Metastatic Transformation in hTERT-Immortalized Basal-Like Human Mammary Epithelial Cells.

Triple negative, basal-like breast cancer is highly aggressive and has a poor prognosis. The molecular mechanisms that drive its progression are elusive and no molecular target has been identified for its prevention or treatment. Here, we demonstrate that triple negative human breast cancer cells and clinical samples show an attenuated transforming growth factor-beta (TGF-β) signaling. Therefore, we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered TGF-β sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative RII (DNRII) receptor of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, co-expression of DNRII and H-ras-V12 rendered HMECs to become highly tumorigenic and metastatic in vivo in comparison with H-ras-V12-transformed HMECs that spontaneously escaped H-ras-V12-induced SLGA. By using microarray analysis, we found that p21 is the major player mediating Ras-induce SLGA and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling is blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-ras-V12-induced SLGA. Our results identifies that autocrine TGF-β signaling is an integral part of cellular antitransformation network by suppressing the expression of a host of genes including p21-regulated genes that mediate oncogene-induced transformation in basal-like breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-05.

P2-01-10: Blockage of Autocrine TGF-b Signaling Reduces Tumorigenecity and Lung Metastasis in a Murine Breast Cancer Cell Line, NMuMG-ST.

Transforming growth factor beta (TGF-b) is known to have a dualistic role in breast cancer cells, acting as a tumor suppressor or a tumor promoter. This dualistic property varies in different cells and at different progression stages. Studies show that autocrine TGF-b is necessary for the growth and survival of MDA-MB-231 cells, and was also known to enhance the survival of MCF-7 cells. However, the role of autocrine TGF-b in spontaneously transformed cells is still less understood. We have retrovirally transduced a dominant negative TGF-b Type II receptor (DN RII) into a spontaneously transformed murine mammary gland epithelial cell line NMuMG-ST (DN RII cells). The expression of DN RII reduces TGF-b sensitivity of NMuMG-ST cells in a gene transcription assay and a growth inhibition assay. Interestingly, the autocrine TGF-b supports the survival of NMuMG-ST cells, although the exogenous TGF-b inhibits the growth of NMuMG-ST cells. Moreover, more apoptosis were observed in the DN RII cells compared to the control cells in both in vivo and in vitro experiment. We found that AKT and ERK pathway mediates autocrine TGF-b9s survival signal. Results of western immunoblot for several stem cell markers and mammosphere formation assay suggest that autocrine TGF-b signaling is essential for the maintenance of stem like cell subpopulation in NMuMG-ST cells. By using immunocytostaining, we found that the DN RII cells expressed more E-cadherin and less Vimentin when compared to the control NMuMG-ST cells, which indicate that blocking autocrine TGF-b signaling pathway could inhibit the process of EMT in NMuMG-ST cells. Notably, when the cells were inoculated orthotopically in nude mouse mammary glands, tumors formed by DN RII cells grew at a similar rate as those formed by control cells, but caused fewer lung metastases. Our finding identified that the autocrine TGF-b signaling is important for the survival and metastatic ability of NMuMG-ST, which could provide an important foundation for further investigation on the role of autocrine TGF-b signaling in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-10.

P1-04-02: In Vitro Qualification and Quantification of Murine Mammary Stem/Progenitor Cells.

Increasing evidence suggest that tumors with a cell origin of more basal or stem-like sources are often highly metastatic and associated with poor prognosis. Identification of the cell of origin thus has important implications for development of new preventive and therapeutic approaches. To identifying the cells of origin of various breast cancers, understanding the normal cellular hierarchy within the breast tissue is an important prerequisite. However, current understanding of normal mammary stem/progenitor cell is limited due to the lack of a robust in vitro assay. The newly developed cell surface markers (CD24 + CD49f hi ) can only be used for low percent of enrichment. The only gold standard assay for functional evaluation of stem cell property is the in vivo transplantation of cleared mammary fat pad assay, yet this assay is expensive, time consuming, and requires highly trained skills, and thus cannot be used routinely for experiments. The goal of our study is thus to develop a robust in vitro assay to qualify and quantify murine mammary stem/progenitor cells. Our rationale lies on our recent findings of in vitro study of different fractions of murine stem/progenitor cells. When we use isolated primary epithelial cells for in vitro mammosphere formation, we found that stem cell enriched fraction and progenitor cell enriched fraction both formed very small mammospheres (≤ 50 μm) and these spheres appear to be of clonal origin (one cell gives rise to one sphere). When we plated these small spheres into 3D extracellular matrix for colony formation, however, they gave rise to two distinct structures: stem-enriched fraction generated predominately solid structure while progenitor-enriched fraction is dominated by hollow structures. Previous studies have linked the solid structure to the basal/myoepithelial lineage and the hollow structure to the luminal lineage. We thus suspected that the small mammospheres derived from the stem or progenitor enriched fractions could be originated from single stem or progenitor cell, and the number of spheres could indicate the number of original stem/progenitor cells within these enriched fractions. Subsequent in vivo transplantation with single sphere or single solid structure cultured in 3D extracellular matrix derived from green fluorescent protein transgenic mice proved that single sphere or 3D solid structure can repopulate the gland-free mammary fat pad. To conclude, the in vitro mammosphere formation in combination with subsequent differentiation in 3D extracellular matrix can be used as a robust in vitro assay for qualification and quantification of murine mammary stem/progenitor cells. This in vitro assay will greatly facilitate our understanding of genes regulate stem cell self-renewal, proliferation, differentiation as well as mechanisms keeping them at quiescent state within the niche. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-02.

P2-01-12: Determining the Molecular Signature That Drives Breast Cancer-Induced Brain Metastasis in a Mouse Xenograft Model.

Breast cancer (BCa) is the most common malignant disease in women in U.S. and the mortality in BCa patients is due to the metastasis of the disease from primary site. Brain metastasis accounts for nearly 20% of all metastases in BCa. It is the most feared complication of BCa because of very few effective treatment regimens available, leading to lower survival rate in patients. The exact molecular mechanism for metastases of BCa into brain is unknown. Rodent model systems have been reported for brain metastasis in BCa but the current models for brain metastasis have limitations. Therefore, there is a need of efficient model system that can significantly contribute towards our understanding of different factors from both host and tumor leading to brain metastasis. Previously we reported isolation and characterization of estrogen independent B6TC cells that are derived from the stable spontaneous fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in mouse bone marrow microenvironment. This chimeric B6TC has propensity to metastasize to brain when inoculated through intracardiac (I.C.) route, and express stem cell-like features. In the present study using B6TC, we have developed an efficient and novel mouse model for studying BCa-induced brain metastasis. We have generated three cell lines from B6TC through three successive rounds of inoculation in mouse and subsequent isolation of brain metastatic cells. Each round of selection enhanced the brain metastatic propensity. An initial microarray analysis identified genes like MMP1, HB-EGF, ST3GAL1, PTGS2, ITGA3, and CXCR4, which showed significant up-regulation in B6TC compared to its parental metastatic MDA-MB-231 or nonaggressive ZR-75-1 cells. These genes are implicated in metastasis regulation. A second round of RNA microarray was performed with three sublines of B6TC with successively enhanced brain metastatic propensity to identify unique potential brain metastatic genes showing gradual up or down-regulation over generations. From analysis of the gene expression profiles, apart from potent brain metastatic genes detected earlier, we also identified some molecular pathways, including TGF beta signaling pathway that are associated with enhanced brain metastasis. The B6TC model is novel for studying the molecular mechanism of brain metastasis, as in this model, apart from experimental metastasis in brain through I.C route, cells show spontaneous metastases to the brain from the primary tumor and this unique feature will enable us to study the mechanisms of the early steps of brain metastasis progression. Further analyses to find out common miRNAs that are over or under-expressed in the sublines over successive generations and determination of gene targets of miRNA are underway. This study will not only provide valuable insight into molecular mechanism of BCa-induced brain metastasis but also lay the foundation to identify novel prognostic and therapeutic markers of brain metastases, leading ultimately to the discovery of novel molecularly targeted drugs to prevent and eradicate BCa metastasis initiation, progression and recurrence. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-12.

P2-01-07: Proto-Oncogene PELP1 Promotes Metastasis through miR-200 Dependent Pathway.

Background: Endocrine therapy is the most important component of adjuvant therapy for patients with early stage estrogen receptor (ER)-positive breast cancer. Despite positive effects of hormonal therapy, initial or acquired resistance to endocrine therapies frequently occurs and tumor recurs as advanced metastatic disease. Accumulating evidence suggests that ER-coregulators play an essential role in cancer progression and that metastatic tumors have increased expression of coregulators. Proline glutamic acid rich protein (PELP1) is an ER coregulator, its expression is upregulated during breast cancer progression to metastasis. PELP1 is an independent prognostic predictor of shorter breast cancer specific survival and its elevated expression positively associates with markers of poor outcome. The objective of this study is to examine the mechanism and significance of PELP1 mediated signaling in breast cancer metastasis. Methods: To examine the significance of PELP1 in metastasis, we have used two ER+Ve (ZR75, MCF7) and one ER-ve MDA-MB 231 models cell that either stably express PELP1 or PELP1 shRNA. MCF7 and ZR75 cells were used as controls. Role of PELP1 on metastasis was studied using Boyden chamber, wound healing, invasion, MMP and reporter gene assays. Epithelial to Mesenchymal Transition (EMT) real time qPCR Array (Super array) was used to identify PELP1 target genes. Whole genome based microarray analysis was performed to identify PELP1 regulated microRNAs. miRNA mimetics and antamiRs were used to establish the mechanism. Nude mice based assays were performed to study the role of PELP1 on in vivo metastasis. Results: Boyden chamber and wound healing assays showed PELP1 down regulation substantially affect migration of both ER+ve and ER-ve cells and showed alterations in the expression of the EMT markers. Focused microarray studies identified PELP1 modulate expression of eight genes involved in the EMT (including Snail, Twist, ZEB1 and MMPs). In xenograft assays, overexpression of PELP1 in non-metastatic cells increases their propensity for metastasis in vivo, while PELP1 knockdown in metastatic model cells decreased in vivo metastatic potential. Whole genome microRNA array analysis revealed that miR 200a and miR141 were upregulated in cells expressing PELP1-shRNA compared to control shRNA expressing cells. Accordingly, over expression of PELP1 in low metastatic model cells decreased expression of miR200a and miR141. Mechanistic studies showed PELP1 down regulate expression of metastasis suppressive microRNAs (miR200a and miR141) by promoting chromatin modifications. Ectopic expression of miR200a and miR141 mimetic decreased PELP1 mediated metastatic functions. Conclusions: These results suggest that PELP1 play a role in breast cancer metastasis by promoting cell motility/EMT by modulating microRNA expression and blockage of PELP1 axis has potential to reduce metastasis potential of breast cancer cells. Understanding how NR coregulator PELP1 plays a role in metastasis will be useful in maximizing treatment opportunities for metastatic breast cancer. This study is funded by DOD grant. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-07.

Abstract P4-06-01: Significance and Therapeutic Potential of PELP1 in ER-Negative Breast Cancers

Background: During the past 20 years, studies have extensively focused on the role of estrogen receptor (ER) and progesterone receptor (PR) in breast cancer. Even through ER and PR explain the biology of ER-positive tumors, it remain unknown as to what drives ER-negative metastatic tumors. Recent advances implicated potential importance of several additional nuclear receptor (NRs) including ERRα, AR, GR, and PPAR in breast cancer. NR action is complex, requires functional interactions with coregulators, and deregulation of coregulators occur during cancer progression. As a modulator of multiple NR functions, coregulators are likely to play a role in breast cancer progression to metastasis. Recent studies indicated that metastatic tumors have increased expression of corgulators. Proline glutamic acid rich protein (PELP1) is a NR coregulator, and its expression is upregulated during breast cancer progression. The objective of this study is to examine whether proto-oncogene PELP1 contributes to metastatic potential of ER-negative breast cancer cells and to test whether blocking of PELP1 signaling axis will have therapeutic effect. Methods: We have used two ER-negative model cells; (1) MDA-MB231 cells that facilitate study of bone and lungs metastasis of breast cancer cells using Nude mice models, (2) 4T1 cells, a clinically relevant model of spontaneous breast cancer metastasis that facilitate tumor growth studies using syngenic mice. To establish the significance of PELP1 axis, we have established clones stably expressing PELP1-shRNA (MDA-MB231- PELP1 shRNA, 4T1-PELP1 shRNA). Proliferation was measured using CellTiter-Glo assays. Role of PELP1 on metastasis was studied using Boyden chamber, wound healing, invasion and MMP assays. Epithelial to Mesenchymal Transition (EMT) real time qPCR Array (Super array) was used to identify PELP1 target genes and targets were validated using ChIP assays. Nude mice based assays were performed to study the role of PELP1 on in vivo metastasis. Results: MDA-MB231 and 4T1 cells expressing PELP1-shRNA showed decreased PELP1 expression (∼75% of endogenous levels) and exhibited decreased propensity to proliferate in in vitro growth assays. Boyden chamber and wound healing assays showed PELP1 down regulation substantially affect migratory potential of MDA-MB231 and 4T1 cells. PELP1 shRNA model cells showed alterations in the expression of the EMT markers. EMT array studies identified eight genes involved in the EMT (including MMPs, E-cadherin, MTA1) as PELP1 potential target genes and ChIP studies showed PELP1 recruitment to these gene promoters. Overexpression of PELP1 in nonmetastatic cells increases their propensity for metastasis in vivo, while, PELP1 knockdown in metastatic model cells decreased their metastatic potential. Nanopartiles delivering PELP1-siRNA significantly affected the growth and metastatic potential of ER-ve cells. Conclusions: These results suggest that PELP1 play a role in ER-ve breast cancer metastasis by promoting cell motility and EMTand blockage of PELP1 axis reduces metastasis potential of ER-negative breast cancer cells. Understanding how NR coregulator PELP1play a role in metastasis will be useful in maximizing treatment opportunities for metastatic breast cancer. This study is funded by Komen grant KG091267. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-01.

Abstract P5-03-01: Role of Aromatase and Its Inhibitor in Breast Cancer-Induced Tumorigenesis and Bone Metastasis

Aromatase (Aro) is the rate-limiting enzyme that catalyzes the final step in estrogen (E2) biosynthesis. An important strategy to treat hormone-dependent BCa is suppression of estrogen receptor (ER) action by antiestrogens or aromatase inhibitors (AI). Letrozole is a very specific and potent AI. In postmenopausal women, the ovaries cease to make E2 but concentration of E2 in their BCa tissue are maintained at a certain level for survival and proliferation of BCa cells, which is dependent on local E2 formation catalyzed by Aro. Although BCa cells have been shown to express Aro, the local E2 is largely produced by adipose stromal cells in the breast. This raises the question of how ER positive (ER+) metastatic BCa cells survive after they enter blood circulation, where E2 level is very low. We cultured human ER+ BCa CAMA-1, Aro-transfected MCF-7 and ZR-75-1 (ZR) cells in suspension to mimic circulating BCa cells. Interestingly, suspension culture increased Aro expression, suggesting circulating ER+ BCa may up-regulate intracrine E2 activity for survival after leaving the E2-rich adipose stroma at primary site. The expression of Aro also enhances cell proliferation and supplementation of testosterone (T), the substrate of Aro, stimulates this proliferation further. Notably, while these cells show an increased rate of apoptosis in suspension than in adherent culture, addition of T in suspension culture significantly suppressed the rate of apoptosis and addition of letrozole blocked the T-induced cell survival in suspension culture. To investigate the importance of intracrine E2 in promoting tumorigenesis and metastasis, we implanted Aro-expressing ZR cells orthotopically and intracardiacally (I.C.) into female athymic mice; vector-transfected ZR cells were used as control. While control ZR cells were incapable of forming tumors without E2 supplementation, Aro-expressing cells generated orthotopic tumors with no E2 supplementation after 3-weeks of inoculation. More interestingly, mice with I.C. inoculated Aro-expressing cells also presented distant bone metastasis in the mandible and tibiae/femora after 2-weeks of inoculation, detected by whole mouse fluorescence and bioluminescence imaging as the cells were stably transfected with a luciferase and GFP expression vector. To determine whether growth of orthotopic tumors can be inhibited by systemic administration of an AI, we treated the mice in one group with letrozole at 10 mg/mouse/day and the other group with the vehicle as control after the average tumor volume reached 150 mm3. After 3-weeks, the tumor burden in the letrozole treatment group reduced significantly while tumor burden in the control group increased continuously. Our studies show that suspension culture increases expression of Aro mRNA in several ER+ BCa cell lines, which likely results in increased intracrine E2 signaling and contributes to the survival of these BCa cellsin suspension. This provides a mechanistic insight into how ER+ BCa cells may survive the low E2 condition in circulation and subsequently induce distant metastasis as observed in the I.C. model. Our study provides an important foundation for future investigation on how hormone-dependent BCa cells up-regulate Aro expression in circulation and induce bone metastasis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-03-01.