Metastasis Of ER Research Articles

CRABP2 regulates invasion and metastasis of breast cancer through hippo pathway dependent on ER status

BackgroundTriple Negative Breast cancer (TNBC) is incurable cancer with higher rates of relapse and shorter overall survival compared with other subtypes of breast cancer. Cellular retinoic acid binding protein 2 (CRABP2) belongs to fatty acid binding protein (FABP) family which binds with all-trans retinoic acid (RA). Previous studies from the database have reported the patients with high expression of CRABP2 showed different prognosis in ER+ and ER− breast cancer. However, its biological role and exact mechanism in breast cancer remain unknown. This aim of this study was to explore how CRABP2 regulated invasion and metastasis based on the estrogen receptor-α (herein called ER) status in breast cancer.MethodsImmunohistochemical staining method was used to analyze the expression of CRABP2 in human breast cancer tissues. Lentivirus vector-based shRNA technique was used to test the functional relevance of CRABP2 knockdown in breast tumors. Tail vein injection model was used to examine the lung metastasis. Co-immunoprecipitation, Western blotting, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were conducted to investigate the underlying mechanism that influenced the ER to the regulation of CRABP2 to Lats1.ResultsWe observed that knockdown of CRABP2 promotes EMT, invasion and metastasis of ER+ breast cancer cells in vitro and in vivo, whereas overexpression of CRABP2 yields the reverse results. In ER+ mammary cancer cells, the interaction of CRABP2 and Lats1 suppress the ubiquitination of Lats1 to activate Hippo pathway to inhibit the invasion and metastasis of ER+ mammary cancer. However, in ER− mammary cancer cells, the interaction of CRABP2 and Lats1 promote the ubiquitination of Lats1 to inactivate Hippo pathway to promote the invasion and metastasis of ER− mammary cancer.ConclusionsOur findings indicate that CRABP2 can suppress invasion and metastasis of ER+ breast cancer and promote invasion and metastasis of ER− breast cancer by regulating the stability of Lats1 in vitro and in vivo, and it provides new ideas for breast cancer therapy.

Abstract 5200: High CD73 expression, regulated by estrogen signaling, associates with cancer aggressiveness in estrogen receptor (+) breast cancer

Abstract Background: CD73, a cell surface enzyme, catalyzes the generation of adenosine from ATP and ADP in the tumor microenvironment along with CD39. Accumulated extracellular adenosine functions as immune-suppressor, and also binds to adenosine receptors which promotes angiogenesis and cell proliferation that results in accelerate cancer progression. However, the clinical significance and molecular function of CD73 expression in breast cancer remains unclear. Methods: Utilizing breast cancer cohorts of TCGA and GEO, clinical significance and underlying mechanisms were investigated. Molecular experiments were carried out in MCF7 cells, ER(+) breast cancer cell line. Results: In TCGA cohort, CD73 expression level was significantly lower in ER(+) breast cancers compared to ER(-) tumors. Higher CD73 expression was associated with worse overall survival in ER(+) tumors (p=0.003), but not in ER(-) tumors. Gene Set Enrichment Analysis revealed that estrogen response gene sets (Early; p=0.043, Late; p=0.021) were significantly enriched in CD73 low expressing ER(+) tumors, suggesting estrogen signaling may repress CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, whereas tamoxifen treatment enhanced expression of the genes. These findings suggest that CD73 and CD39 gene expression is suppressed by estrogen signaling, whereas binding of ER antagonists such as tamoxifen can remove the repressive effect on gene expression. On the other hand, epithelial-mesenchymal transition (EMT) (p<0.001) and angiogenesis (p<0.001) gene sets were significantly enriched in CD73 high expressing ER(+) tumors. CIBERSORT demonstrated that CD73 high expressing ER(+) tumors have less infiltrating CD8(+) T cells, memory B cells and plasma cells, implying that CD73 high expressing tumors have immune suppressive environment, which is in agreement with the notion that CD73 high tumors are immunosuppressive. Finally, we found that CD73 expression was significantly elevated post-chemotherapy compared to tumors prior to the treatment (p=0.007), and CD73 high expression patients showed worse relapse-free survival in neoadjuvant chemotherapy patients cohort (p=0.003). Conclusion: Molecular studies revealed that CD73 expression is regulated by estrogen signaling. Increased expression of CD73 significantly correlates with worse outcomes in ER(+) breast cancer patients. This may be due to upregulated pro-metastatic gene signatures such as EMT and angiogenesis as well as less infiltration of anti-cancer immune cells by adenosine generated by CD73 in the tumor microenvironment. Our data reveals an intriguing mechanism which may be responsible for recurrence and metastasis of ER(+) breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Eriko Katsuta, Vivek Anand, Li Yan, Subhamoy Dasgupta, Kazuaki Takabe. High CD73 expression, regulated by estrogen signaling, associates with cancer aggressiveness in estrogen receptor (+) breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5200.

Abstract PD7-09: Lasofoxifene decreases breast cancer lung and liver metastasis in a mammary intraductal (MIND) xenograft model of mutant ERα+ breast cancer

Abstract The standard of care for early postmenopausal ERα+ breast cancer patients is adjuvant endocrine therapy, typically an aromatase inhibitor or tamoxifen, and endocrine therapy with or without a CDK 4/6 inhibitor in the metastatic setting. However, a number of these patients are not sensitive to endocrine therapy or experience breast cancer recurrence 10 to 15 years after endocrine treatment, and all eventually progress in the metastatic setting. Metastasis of ER+ therapy-resistant breast cancer correlates with the acquisition of ESR1 activating mutations in up to 40% or more of affected patients. The two most common ERα mutations are Y537S and D538G, both of which confer ERα constitutive activity. Lasofoxifene is a SERM originally developed to treat vulvovaginal atrophy and osteoporosis. In this study, we analyzed the efficacy of lasofoxifene in the treatment of MCF7 tumor explant models that were engineered with CRISPR/Cas9 to express Y537S or D538G ERα. To better mimic the natural micro-environment of infiltrating ductal breast cancer, we used the mammary intraductal mouse model (MIND), in which tumor cells are introduced into the ducts via the nipple. Three MCF7 cell variants, MCF7 WT, MCF7 Y537S and MCF7 D538G were injected into the mammary ducts of NSG mice. Prior to injection, cells were labeled with a luciferase reporter to monitor tumor growth in vivo using a Xenogen IVIS imager. Mice were treated with three different doses of lasofoxifene (1, 5 and 10mg/kg; 5days/wk SQ), vehicle, or the SERD, fulvestrant, (5 mg SQ once per week = 250 mg/kg). After 70 days of treatment, primary tumor growth, as measured by endpoint tumor weight, of MCF7 WT, D538G and Y537S explants was significantly inhibited versus vehicle by 10 mg/kg lasofoxifene and fulvestrant. Compared to fulvestrant, lasofoxifene was significantly more effective at 5 and 10 mg/kg for the MCF7 Y537S and D538G tumors. Notably, the two MCF7 mutants metastasized to the lung and liver, whereas WT MCF-7 cells were only very weakly metastatic by the end of the study. Lasofoxifene significantly inhibited the metastasis of both MCF7 Y537S and D538G to the lungs and liver at 5 and 10 mg/ml. In contrast, fulvestrant only inhibited metastasis of the MCF7 D538G mutant to both organs. In a follow-up combination therapy study of MCF-7 WT and Y537S tumor explants, in which mice were co-treated with either fulvestrant and palbociclib or lasofoxifene and palbociclib, lasofoxifene plus palbociclib demonstrated a better outcome than fulvestrant and palbociclib in terms of primary tumor weight inhibition. Results for lung and liver metastasis inhibition are forthcoming. In conclusion, these data suggest that lasofoxifene may have the potential to be utilized as a treatment for metastatic breast cancers, including those that express constitutively active ERα mutations, including D538G and Y537S, and warrants further study. Citation Format: Laine M, Greene M, Chang Y-F, Phung L, Hiipakka R, Komm B, Greene GL. Lasofoxifene decreases breast cancer lung and liver metastasis in a mammary intraductal (MIND) xenograft model of mutant ERα+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-09.

Abstract 3845: 24R,25-Dihydroxyvitamin D3 reduces tumor burden and metastasis in ER+ breast cancer in vivo

Abstract The purpose of this study was to investigate the effects of 24R,25-dihydroxyvitamin D3 (24,25), on tumor growth and metastasis in an in vivo estrogen receptor positive (ER+) orthotopic breast cancer model. 1α,25(OH)2D3 has been shown to have pro-apoptotic effects on breast cancer both in vitro and in vivo. 24,25, a naturally occurring metabolite, is present in human serum at concentrations up to 10 times higher than 1,25. Moreover, 1,25 regulates production of 24,25 by stimulating expression of the 24-hydroxylase. 6-8 week old female nod scid gamma IL2R mice were kept on an ad libitum vitamin D deficient diet. 15 days before surgery, mice were implanted with 1.7mg 0-order release estrogen (E2) pellets. In the first experiment, mice were given a fast-release pellet (45 days). In the second experiment, mice were given a slow-release (90 day) pellet. After 15 days of acclimation (day 0), mice were injected in the T4 mammary fat pad with 1M fluorescently labeled MCF7 cells (an ER+ breast cancer cell line) and cells in 50:50 PBS:Matrigel solution. Tumors were measured once a week starting at day 14 and tracked throughout the course of the study. On day 30, 24,25 treatment was initiated. Each experiment was divided into three groups: 0 ng 24,25, 25 ng 24,25, or 100 ng 24,25 per injection, with n=6-8 mice per group. Treatments were administered three times a week in a 50µL intraperitoneal injection. On day 70, mice were harvested and assessed for tumor burden and metastasis by fluorescence detection microscopy. Fluorescent tissues were marked ‘metastasis positive' and harvested for future immunohistochemical analysis. Mice that were given 24,25 three times weekly showed decreased tumor burden and metastasis as compared to controls. In the first experiment (45 day E2), 25ng and 100ng 24,25 treated mice had significantly lower relative tumor volumes than controls beginning on day 70. These 24,25 treated mice also showed significantly lower rates of left axial lymph node metastasis, and had statistically longer survival rates. The second experiment (90 day E2) had similar results. Mice treated with 25ng and 100ng 24,25 had decreased tumor burden as compared to 0ng controls on day 70. Mice given 24,25 had decreased lung metastasis; however, mice treated with 100ng 24,25 had increased rates of right axial lymph node metastasis. Altogether, mice in both groups that were treated with 100ng of 24,25 had decreased rates of left axial lymph node metastasis and lung metastasis as compared to controls, and statistically longer survival rates. Thus 24,25 could play a role in 1,25's anti-apoptotic effect on breast cancer, potentially via upregulation of 24,25. Citation Format: Anjali Verma, David Joshua Cohen, Chandana Muktipaty, Barbara D. Boyan, Zvi Schwartz. 24R,25-Dihydroxyvitamin D3 reduces tumor burden and metastasis in ER+ breast cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3845.

Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis

BackgroundThe majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown.MethodsWe used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses.ResultsKnockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ∆Np63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2− tumors, was associated with poor outcome.ConclusionThese studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.

Abstract P1-09-12: mTORC1 is responsible for metastasis of ER+ breast cancer downstream of RON tyrosine kinase

Abstract Metastatic breast cancer is one of the major challenges of tumor biology, remaining the underlying cause of death in the vast majority of breast cancer patients. Despite advances in treatment strategies, metastatic breast cancer is still considered incurable and mostly resistant to the current treatment options. There is, therefore, a clear need for development of novel targeted therapeutic agents, which must be built on a foundation of a deeper understanding of the molecular mechanisms responsible for development of metastasis. It is well accepted that aberrant expression of the RON receptor tyrosine kinase in breast cancer contributes to development of metastasis, and is associated with poor patient prognosis. Elevated expression of RON has been reported in about 50% of primary breast cancer samples. However, in later metastatic stage, RON is overexpressed in 100% of breast cancer samples, pointing toward its important role in inducing metastasis. RON is known to activate multiple signaling pathways, including MAPK, PI3K, Src, phospholipase C-γ and β-catenin. Although some of these pathways have been linked to specific biological outcomes in vitro, contributions of each signal transduction pathway to cancer progression and metastasis are still poorly defined. To address this critical question, we used a combination of inhibitory and mutational strategies. We engineered T47D cells to conditionally overexpress RON upon addition of doxycycline, enabling us to achieve titratable levels of RON. For inhibition of RON signaling, we used a highly selective RON inhibitor, ASLAN002. To obtain a broader knowledge about signaling networks downstream of RON, we used a high-throughput functional proteomics assay, Reverse Phase Protein Array (RPPA). RPPA analysis showed robust phosphorylation of ribosomal protein S6 (rpS6) upon RON activation, which could be reversed by RON inhibitor. We were then able to dissect the components of the pathway responsible for phosphorylation of rpS6 using inhibitors against different potential kinases. These analyses revealed that mTORC1 is the main kinase responsible for profound phosphorylation of rpS6 downstream of RON, which itself is fed dominantly by PI3K rather than MAPK. To validate the PI3K/mTORC1/p70S6K axis as the main feeder of rpS6, we took advantage of a mutational approach to modulate RON signaling to specific pathways. Biochemical analysis of various mutants in T47D cells showed that they signal differentially, with mutant A strongly signaling through PI3K/mTORC1. To assess the importance of PI3K/mTORC1 in RON-mediated metastasis, we conducted in vivo studies. Our results indicated that mutant A is the highest metastatic mutant, inducing metastasis in 100% of the mice studied. To examine whether mTORC1 is critical for induction of metastasis downstream of RON, we treated mice with mTORC1 inhibitor, everolimus, and monitored metastasis. Our data revealed that mTORC1 inhibition could dramatically shrink metastatic lesions, before resistance occurs. We report a dissected analysis of the RON signaling pathway responsible for induction of metastasis in ER+ breast cancer. Our data strongly indicate that RON dominantly signals through PI3K/mTORC1/p70S6K/rpS6, and that this pathway is important in inducing metastasis. Citation Format: Faham N, welm A. mTORC1 is responsible for metastasis of ER+ breast cancer downstream of RON tyrosine kinase [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-12.

Androgen receptor/let-7a signaling regulates breast tumor-initiating cells.

Androgen receptor (AR) is an important transcriptional factor, which is frequently expressed in invasive breast cancer and correlates patients’ prognosis. Our previous results indicate AR activation may increase let-7a expression in breast cancer cells, while let-7, a tumor suppressor, is reported to inhibit breast tumor-initiating cells (T-IC). The study aims to explore the effects of AR/let-7a signaling on breast T-IC and its regulatory mechanism. The results revealed that the expression of AR was significantly associated with let-7a and CD44+/24-/low especially in estrogen receptor positive (ER+) breast cancer tissues. The expression of AR and let-7a indicated better outcome, while patients with CD44+/24-/low phenotype had worse prognosis. AR activation induced by dihydrotestosterone (DHT) prevented cells proliferation, migration, invasion and self-renewal capacities in ER+ breast cancer cells, via transcriptional up-regulation of let-7a. In addition, AR could inhibit tumorigenesis and metastasis of ER+ breast cancer cells in the serial xenotranplanted animal models. Our data suggested that AR/let-7a signaling could inhibit the biological behavior of tumor-initiating cells (T-IC) in ER+AR+ breast cancers, which might become a new therapeutic target.

Abstract 2365: Dual inhibition of the PI3k/Akt and MEK5/ERK5 pathways in tamoxifen resistant breast cancer

Abstract Approximately 70% of breast cancers are considered estrogen receptor alpha (ER) and/or progesterone receptor (PR) positive, and their growth is primarily driven by the hormone estrogen. Initially these cancers are responsive to endocrine therapies such as tamoxifen. However, over time patients become resistant to conventional endocrine therapy and treatment becomes more difficult. Tamoxifen resistance occurs due to several mechanisms: loss of ER expression, gain of function mutations, pharmacological tolerance, alterations of co-activators, and ligand independent activation of ER by various kinase cascades. Aberrations in the Phosphoinositide-3-kinase (PI3K) and Mitogen Activated Protein Kinase (MAPK) pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. The crosstalk between PI3K/Akt and MEK1/2/ERK1/2 has been characterized in several systems. However, new evidence suggests that MEK5/ERK5, a member of the MAPK family, is a key component in the proliferation and survival of therapy resistant cancers. MEK5/ERK5 has been shown to promote ER alpha driven transcription in ER+ breast cancers and actin reorganization and metastasis in ER- breast cancers. Furthermore, MEK5/ERK5 promotes hormone independent tumorigenesis in breast cancer. Our lab has previously investigated these pathways in hormone independent breast cancers, TNBC (triple negative breast cancer). Our previous results indicate that combinations of PI3k/Akt and MEK5/ERK5 blockade are promising because they inhibit both the pro-proliferative and pro-metastatic pathways in TNBC. Additionally, PI3k/Akt and MEK5/ERK5 inhibition was shown synergistically reduce viability in a diverse panel of cell lines (TNBC, ER+, and Triple Positive). Based on these initial results we hypothesize that PI3k/Akt and MEK5/ERK5 inhibition decreases viability via a hormone independent mechanism: perhaps by reducing the cytosolic sequestration of Bad. In this study we investigate the utility PI3k/Akt and MEK5/ERK5 inhibition in a MCF-7 TamR cell line. The goal of our study is to elucidate the roles of PI3k/Akt/mTOR and MEK5/ERK5 in endocrine resistant breast cancer and broaden the scope of a dual inhibition strategy. Citation Format: Thomas D. Wright, Mahmud Hasan, Paula Witt-Enderby, Jane Cavanaugh. Dual inhibition of the PI3k/Akt and MEK5/ERK5 pathways in tamoxifen resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2365. doi:10.1158/1538-7445.AM2017-2365

Abstract 4410: ZEB2 drives cell motility and metastasis in ER+ breast cancer cells through a novel, E-cadherin independent pathway

Abstract Breast cancer is the most commonly diagnosed cancer in women, with the second highest mortality rate. The overwhelming majority of breast cancer deaths are a direct result of distant metastatic spread to vital organs such as lung, bone, and brain. The zinc finger transcription factor ZEB2 has been implicated as a driver of cancer cell motility, tissue invasion and metastasis in a number of diverse malignancies, but its role in breast cancer is not completely understood. We chose to examine the effects of direct overexpression of ZEB2 in the MCF-7 cell line, a luminal and estrogen receptor positive (ER+) derived breast cancer line. MCF-7-ZEB2 cells demonstrated significantly increased migration and invasion as well as an altered morphology in vitro compared to that of vector. Additionally, MCF-7-ZEB2 cells exhibited increased lung metastasis in vivo when implanted in an orthotopic xenograft mouse model. ZEB2 function in metastasis has canonically been attributed to transcriptional repression of the cell junction protein E-Cadherin. Here we establish that in ER+ breast cancer cells, ZEB2 fails to repress E-cadherin and promotes cell motility and metastasis through the induction of an E-cadherin independent signaling cascade. Next generation RNA sequencing analysis of the MCF-7-ZEB2 cells compared to vector revealed alteration of the MAPK signaling cascade, evidenced by enhanced expression of key motility and MAPK associated genes, including PLAU, EGF, ACTA2, and MMP9. Pharmacological inhibition of MAPK pathway completely abrogated ZEB2 induced migration, cell morphology changes and expression of target motility genes, confirming a necessary role for MAPK signaling in ZEB2-driven cell motility in ER+ breast cancer. Together these results indicate that the ZEB2 transcription factor drives motility in breast cancer cells through a novel MAPK dependent pathway, warranting further investigation into the mechanisms involved in ZEB2 action. Elucidating the pathways involved in ZEB2 function which are specific to ER+ breast cancer is an important step in understanding the processes underlying metastasis and has the potential to yield new therapeutic targets. Citation Format: Hope E. Burks, Lyndsay V. Rhodes, Elizabeth C. Martin, Van T. Hoang, Steven Elliott, Melody Badoo, Theresa Phamduy, Aaron Buechlein, Douglas Rusch, Douglas Chrisey, Erik Flemington, Kenneth Nephew, Bridgette Collins-Burow, Matthew E. Burow. ZEB2 drives cell motility and metastasis in ER+ breast cancer cells through a novel, E-cadherin independent pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4410.

Gene expression alterations associated with outcome in aromatase inhibitor-treated ER+ early-stage breast cancer patients.

Aromatase inhibitors (AI), either alone or together with chemotherapy, have become the standard adjuvant treatment for postmenopausal, estrogen receptor-positive (ER+) breast cancer. Although AIs improve overall survival, resistance is still a major clinical problem, thus additional biomarkers predictive of outcome of ER+breast cancer patients treated with AIs are needed. Global gene expression analysis was performed on ER+ primary breast cancers from patients treated with adjuvant AI monotherapy; half experienced recurrence (median follow-up 6.7years). Gene expression alterations were validated by qRT-PCR, and functional studies evaluating the effect of siRNA-mediated gene knockdown on cell growth were performed. Twenty-six genes, including TFF3, DACH1, RGS5, and GHR, were shown to exhibit altered expression in tumors from patients with recurrence versus non-recurrent (fold change ≥1.5, p<0.05), and the gene expression alterations were confirmed using qRT-PCR. Ten of these 26 genes could be linked in a network associated with cellular proliferation, growth, and development. TFF3, which encodes for trefoil factor 3 and is an estrogen-responsive oncogene shown to play a functional role in tamoxifen resistance and metastasis of ER+ breast cancer, was also shown to be upregulated in an AI-resistant cell line model, and reduction of TFF3 levels using TFF3-specific siRNAs decreased the growth of both the AI-resistant and -sensitive parental cell lines. Moreover, overexpression of TFF3 in parental AI-sensitive MCF-7/S0.5 cells resulted in reduced sensitivity to the AI exemestane, whereas TFF3 overexpression had no effect on growth in the absence of exemestane, indicating that TFF3 mediates growth and survival signals that abrogate the growth inhibitory effect of exemestane. We identified a panel of 26 genes exhibiting altered expression associated with disease recurrence in patients treated with adjuvant AI monotherapy, including TFF3, which was shown to exhibit a growth- and survival-promoting effect in the context of AI treatment.

Abstract 1034: ZEB2 promotes cell motility and metastasis in ER+ breast cancer cells

Abstract While the precise molecular mechanisms underlying metastasis remain unclear, epithelial-to-mesenchymal transition (EMT), the loss of an epithelial cell phenotype and acquisition of a mesenchymal cell phenotype, has been implicated in cancer cell invasion and dissemination. The ZEB family of transcription factors, which includes ZEB1 and ZEB2, has been demonstrated to mediate this transition by downregulating the expression of genes associated with an epithelial phenotype. We sought to investigate the effects of direct ZEB family overexpression on EMT in estrogen receptor-positive (ER+) breast cancer cell systems. We overexpressed ZEB1 or ZEB2 in the epithelial, ER+, luminal A breast cancer cell lines MCF-7 and ZR75. Overexpression of individual ZEB1 and ZEB2 levels were confirmed and localization of the ZEB factors to the nucleus was confirmed by confocal microscopy in both cell lines. ZEB2 overexpressing cells, but not ZEB1 overexpressing cells, showed increased migration and invasion in vitro compared to the vector control in both MCF-7 and ZR75 cell lines, suggesting differential function of the two ZEB family members. Additionally, MCF-7-ZEB2 xenografts exhibited increased lung metastasis compared to MCF-7-vector cells. To elucidate the effects of ZEB on our ER+ cell line we performed next generation deep sequencing on MCF-7 -vector, ZEB1 and ZEB2 overexpressing cells. Analysis of total gene regulation using the NCI Pathway Interaction Database demonstrated an increase in genes associated with RhoA activity, an important mediator of cell motility, in ZEB2 overexpressing cells. However, the ZEB overexpressing cells show no change in morphology and canonical EMT markers remained unchanged between cell lines, suggesting a potential post-translational modification affecting ZEB1 and ZEB2 function. Together these results indicate that ZEB factors drive motility in breast cancer cells but are incapable of promoting a complete EMT in ER+ cells, warranting further investigation into the mechanisms involved in ZEB action. Elucidating the pathways involved in ZEB family function is an important step in understanding the mechanisms underlying metastasis and has the potential to yield new therapeutic targets. Citation Format: Hope E. Burks, Lyndsay Rhodes, Elizabeth Martin, Theresa Phamduy, Steven Elliot, Van Hoang, Henry Segar, Aaron Buechlein, Douglas Rusch, Dave Miller, Melody Baddoo, Erik Flemington, Kenneth Nephew, Douglas Chrisey, Bridgette Collins-Burow, Matthew Burow. ZEB2 promotes cell motility and metastasis in ER+ breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1034. doi:10.1158/1538-7445.AM2014-1034

Trefoil factor 3 promotes metastatic seeding and predicts poor survival outcome of patients with mammary carcinoma.

IntroductionRecurrence or early metastasis remains the predominant cause of mortality in patients with estrogen receptor positive (ER+) mammary carcinoma (MC). However, the molecular mechanisms underlying the initial progression of ER+ MC to metastasis remains poorly understood. Trefoil factor 3 (TFF3) is an estrogen-responsive oncogene in MC. Herein, we provide evidence for a functional role of TFF3 in metastatic progression of ER+ MC.MethodsThe association of TFF3 expression with clinicopathological parameters and survival outcome in a cohort of MC patients was assessed by immunohistochemistry. The expression of TFF3 in MCF7 and T47D cells was modulated by forced expression or siRNA-mediated depletion of TFF3. mRNA and protein levels were determined using qPCR and western blot. The functional effect of modulation of TFF3 expression in MC cells was determined in vitro and in vivo. Mechanistic analyses were performed using reporter constructs, modulation of signal transducer and activator of transcription 3 (STAT3) expression, and pharmacological inhibitors against c-SRC and STAT3 activity.ResultsTFF3 protein expression was positively associated with larger tumour size, lymph node metastasis, higher stage, and poor survival outcome. Forced expression of TFF3 in ER+ MC cells stimulated colony scattering, cell adhesion to a Collagen I-coated matrix, colony formation on a Collagen I- or Matrigel-coated matrix, endothelial cell adhesion, and transmigration through an endothelial cell barrier. In vivo, forced expression of TFF3 in MCF7 cells stimulated the formation of metastatic nodules in animal lungs. TFF3 regulation of the mRNA levels of epithelial, mesenchymal, and metastatic-related genes in ER+ MC cells were consistent with the altered cell behaviour. Forced expression of TFF3 in ER+ MC cells stimulated phosphorylation of c-SRC that subsequently increased STAT3 activity, which lead to the downregulation of E-cadherin. siRNA-mediated depletion of TFF3 reduced the invasiveness of ER+ MC cells.ConclusionsTFF3 expression predicts metastasis and poor survival outcome of patients with MC and functionally stimulates cellular invasion and metastasis of ER+ MC cells. Adjuvant functional inhibition of TFF3 may therefore be considered to ameliorate outcome of ER+ MC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0429-3) contains supplementary material, which is available to authorized users.

Abstract P2-11-06: Comparison of PAM50 risk of recurrence (ROR) scores with EndoPredict for predicting risk of distant metastasis in ER+/HER2-, early node-positive breast cancer patients treated with adjuvant chemotherapy - A GEICAM/ 9906 sub-study

Abstract Background: Several prognostic multigene tests are available for the management of breast cancer patients, but so far there is little data available directly comparing the classification performance of different breast cancer tests in the same patient cohort. Here, we compared the prognostic performance of two second generation multigene tests in ER+/HER2- early node-positive breast cancer patients treated with adjuvant chemotherapy followed by endocrine therapy. Patients and methods: The EndoPredict (EP) score and two research laboratory-based versions of the PAM50 risk of recurrence (ROR based on subtype [ROR-S] and based on subtype and proliferation [ROR-P]) score were compared in 536 ER+/HER2- breast cancer patients who participated in the GEICAM/ 9906 trial. Patients had either been treated with fluorouracil, epirubicin, and cyclophosphamide (FEC) or with FEC followed by weekly paclitaxel (FEC-P), and endocrine therapy. ROR and EP scores were combined with clinical information to obtain the following hybrid scores as published before: a) ROR-T score (subtype and tumor size), b) ROR-PT score (subtype, proliferation and tumor size) scores, and c) EPclin score (EP Score plus tumor size and nodal status). Each patient was assigned to risk categories according to pre-specified cut-off levels for the scores. The primary endpoint was distant metastasis. Metastasis rates were estimated using the Kaplan-Meier method. C-index analysis was used to estimate the performance of the different scores. Results: The PAM50 ROR-S and ROR-P scores, and the EP score were prognostic in patients with ER+/HER2- tumors and consistently identified a low-risk group with a particular good outcome (10 year MFS rate - ROR-S: 81.7%, ROR-P: 82.9% and EP: 89.4%). Combining the molecular signatures with clinical information, improved the prognostic performance of all signatures (10 year MFS rate - ROR-T: 83.4%, ROR-PT: 87.3% and EPclin: 100%). All signatures added prognostic information to common clinical parameters. Among the signatures evaluated only EPclin added a significantly improved prediction of distant metastasis to ROR-T (p &amp;lt; 0.001) and ROR-PT (p &amp;lt; 0.001), which may be due to the feature that EPClin includes a term for node status, but which is not included in any ROR-based model. Conclusions: Our study suggests that PAM50 ROR and EP can be used to reliably predict risk of distant metastasis in node-positive ER+/HER2- breast cancer patients treated with chemotherapy. There was no significant difference between the molecular signatures in terms of low risk classification. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-06.

P3-01-04: Differential Impact of Gefitinib and PLX4720 on Proliferation of MCF10A and Isogenic Lines as Measured with a Metastasis Expression Score.

Abstract Background: We previously reported that a published “Metastasis Score” (MS) could be used to evaluate the effects of a PI3K inhibitor (GDC-0941) on mutated human isogenic breast cancer cell lines. MS, based on the expression of14 genes, has been shown to predict distant metastasis in ER(+), node (−), breast cancer. We were interested to determine the impact of gefitinib (EGFR inhibitor) and PLX-4720 (selective inhibitor of BRAF V600E) on MS to assess the applicability of this score to a broader class of targeted agents. In addition, given the cross-talk between metabolism and proliferation, we also profiled genes involved in glycolysis, fatty acid metabolism and oxidative phosphorylation. Methods: Parental MCF10A (WT for all genes) and isogenic lines of MCF10A harboring PI3K (H1047R), p53 null or KRAS(G12V) mutations were cultured overnight in DMEM:F12 media under identical conditions. Cells were then treated with gefitinib, PLX4720, or DMSO (vehicle control) and further incubated for 24hr. Expression analysis was performed by RT-PCR. Results: The MS of the PI3K (H1047R) and p53 null lines was higher than the parental line and lower for the KRAS (G12V) line. Treatment of parental, PI3K and KRAS cell lines with gefitinib resulted in dose-dependent decreases in MS, as reported for GDC-0941, with a higher dose required to inhibit growth of PI3K (H1047R) cells. Treatment of p53 null cells with gefitinib, however, had only a modest effect on MS. In contrast to gefitinib, MS increased with PLX-4720 treatment in all 4 lines; the greatest increase was observed in KRAS (G12V) cells. The expression of metabolic genes differed significantly depending on the oncogenic mutation harbored by the cell line. ACTA2, ACLY, RPIA, KHK, GLS2 were most highly expressed in p53 null but lowest in KRAS (G12V), while GLUT5, PFKFB4, ENO3, SLC27A1, PGK2, GLUT1, TKT were expressed most highly in KRAS (G12V) but lowest in PI3K(H1047R). Upon treatment with gefitinib or PLX-4720, genes that were downregulated in the mutated lines generally showed a dose-dependent increase; those that were upregulated relative to the parental, showed a dose-dependent decrease with treatment. Discussion: The decrease in MS of the parental, PI3K (H1047R) and KRAS (G12V) cell lines treated with gefitinib supports the impact of this EGFR inhibitor on cell proliferation. The modest effect on MS in p53 null cells supports the findings that sensitivity to gefitinib requires active p53 in order to induce apoptosis through a p53-dependent pathway. Increases in MS were observed in all 4 cell lines treated with PLX-4720, with the largest increase observed in KRAS (G12V). The enhanced proliferation of a BRAF WT cell line with a KRAS mutation is consistent with the Ras-dependent nature of this pathway. The metabolic genes showed diverse expression patterns that differed with different oncogenic mutations and likely reflect the multiple mechanisms controlling metabolism in cancer. An improved understanding of the expression of metabolic genes relative to proliferation in cell lines with various oncogenic mutations may provide additional insights into the dysregulation of these cellular processes and the possible role of anti-metabolite intervention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-04.

Abstract LB-203: Aromatase expression promotes breast cancer tumorigenesis and bone metastasis

Abstract Aromatase P450 (CYP19) is the rate-limiting enzyme catalyzing the final step in estrogen biosynthesis, and an important target in breast cancer therapy. In post-menopausal women, ovaries cease to make estrogen, yet the incidence of breast cancer increases with age as aromatase present in breast adipose stroma and epithelium catalyzes local estrogen formation. The circulating estrogen level in postmenopausal women is very low and it reflects the sum of local estrogen biosynthesis in various sites. Although breast cancer cells have been shown to express aromatase prompting the hypothesis that intracrine action of estrogen in breast cancer cells may drive tumor progression, there has been little evidence in addressing the hypothesis. We stably transfected the ER positive (ER+) breast cancer ZR-75-1 cells with an human aromatase expression vector and obtained a few clones expressing a moderately higher level of aromatase in comparison with the control vector-transfected cells. One of the clones called ZR-75-1/Aro/Clone10 (Cl10) was selected for further study due to its increased intracellular estradiol (E2) concentration compared to other clones. The Cl10 cells formed tumors after being xenografted into the inguinal mammary fat pad of female athymic mice while the control cells were incapable of forming tumors without estrogen supplementation. Implantation of testosterone pellet further stimulated the growth of the tumor and a cell line, ZR-75-1/Aro/Clone10/TT1 (TT1) was established from the tumor. Both Cl10 and TT1 cells expressed similar levels of aromatase. Interestingly, the aromatase expression increased when the cells were cultured in suspension suggesting circulating ER+ breast cancer cells may up-regulate intracrine estrogen activity for survival after leaving the estrogen-rich adipose stroma at primary site. To investigate the importance of intracrine estrogen in promoting tumorigenesis and metastasis, we implanted the Cl10 and TT1 cells orthotopically and intracardiacally (I.C.) into female athymic mice. Notably, both cell lines generated orthotopic tumors with no estrogen supplementation after three weeks of inoculation. More interestingly, the mice with I.C. inoculated aromatase-expressing cells presented distant metastasis to bones in mandible and legs after two weeks of inoculation detected by whole mouse fluorescence and bioluminescence imaging as both cells were stably transfected with a luciferase and GFP expression vector. We will determine whether growth of the orthotopic tumors and bone metastases can be inhibited with systemic administration of an aromatase inhibitor and whether bone metastasis is osteoclastic or osteoblastic. Our studies show that a moderate expression of aromatase in breast cancer cells is sufficient to generate intracrine estrogenic action and promote tumorigenesis and skeletal metastasis in ER+ breast cancer cells. These aromatase-expressing models should be useful for future investigation on how ER+ breast cancer cells up-regulate aromatase expression in suspension and induces bone remodeling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-203.