Model Of Breast Cancer Progression Research Articles

Regulation of miRNA-29c and its downstream pathways in preneoplastic progression of triple-negative breast cancer.

Little is understood about the early molecular drivers of triple-negative breast cancer (TNBC), making the identification of women at risk and development of targeted therapy for prevention significant challenges. By sequencing a TNBC cell line-based breast cancer progression model we have found that miRNA-29c is progressively lost during TNBC tumorigenesis. In support of the tumor suppressive role of miRNA 29c, we found that low levels predict poor overall patient survival and, conversely, that ectopic expression of miRNA-29c in preneoplastic cell models inhibits growth. miRNA-29c exerts its growth inhibitory effects through direct binding and regulation of TGFB-induced factor homeobox 2 (TGIF2), CAMP-responsive element binding protein 5 (CREB5), and V-Akt murine thymoma viral oncogene homolog 3 (AKT3). miRNA-29c regulation of these gene targets seems to be functionally relevant, as TGIF2, CREB5, and AKT3 were able to rescue the inhibition of cell proliferation and colony formation caused by ectopic expression of miRNA-29c in preneoplastic cells. AKT3 is an oncogene of known relevance in breast cancer, and as a proof of principle we show that inhibition of phosphoinositide 3-kinase (PI3K) activity, a protein upstream of AKT3, suppressed proliferation in TNBC preneoplastic cells. We explored additional opportunities for prevention of TNBC by studying the regulation of miRNA-29c and identified DNA methylation to have a role in the inhibition of miRNA-29c during TNBC tumorigenesis. Consistent with these observations, we found 5 aza-cytadine to relieve the suppression of miRNA-29c. Together, these results demonstrate that miRNA-29c loss plays a key role in the early development of TNBC.

Abstract B28: Induced expression of Sprouty4 in breast invasive ductal carcinoma cells inhibits ERK MAP kinase and reduces malignant phenotype

Abstract Our previous work has identified increased expression of Sprouty4 in 3D models of breast ductal carcinoma in situ (DCIS). Sprouty4 in other systems has been shown to function as a negative regulator of the mitogen activated protein kinase (MAPK/ERK) pathway. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling in DCIS, and that its loss or reduced expression is one mechanism by which triple-negative lesions progress toward invasive ductal carcinoma (IDC). Using immunohistochemistry our labs have found that Sprouty4 was highly expressed in some human premalignant breast tissue samples, and that this expression was reduced in malignant triple-negative samples. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient over-expression of Sprouty4 reduced both MAPK/ERK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments done in IDC cells overexpressing Sprouty4 revealed relocation of E-cadherin back to the cell surface and the restoration of adherens junctions. To determine if these effects were due to changes in MAPK/ERK signaling, IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug restored an epithelial-like phenotype similar to that produced by Sprouty4 over-expression. From these data we conclude that Sprouty4 may act to control MAPK/ERK signaling in a subset of DCIS, thus limiting the progression of these premalignant breast cancers. Further, if in vivo data correspond with our in vitro work, this may argue for the investigation of MEK inhibitors as an adjunct treatment in triple-negative disease, where options are limited. Citation Format: Ryan M. Jackson, Ethan J. Brock, Seema Shah, Mansoureh Sameni, Bonnie F. Sloane, Quanwen Li, Raymond R. Mattingly. Induced expression of Sprouty4 in breast invasive ductal carcinoma cells inhibits ERK MAP kinase and reduces malignant phenotype. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B28.

Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model.

The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three‐dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non‐malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1–HRAS and UBA2–PDCD2L expressed in‐frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer‐relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 57: Twist1 modifies the metabolic state of breast cancer cells through transcriptional activation of a key regulator of glycolysis

Abstract The shift from a normal to high glycolytic state (Warburg effect) is a central hallmark of almost every cancer and a strong driving force for cancer cell survival and disease progression. Importantly, studies - including our own - have shown that normalization of metabolic state in cancer cells can halt or reverse malignancy. To date, the intrinsic and extrinsic factors that drive metabolic shifts in cancer cells are not entirely clear. A better understanding of how the metabolic state of cancer cells is regulated will give us important tools to inhibit disease progression. We have established a novel model of breast cancer progression consisting of three cancer cell lines established from the primary tumor (TES1) and two sites of metastasis (TES2b and TESPE) of the same patient. We compared gene expression profiles between primary and recurrent tumor cells to identify factors that regulate disease progression and dissemination. Key differences between cells derived from primary and recurrent disease were an activation of genes involved in glycolysis and genes involved in activation and maintenance of the epithelial to mesenchymal transition (EMT) program, a powerful driver of cancer metastasis. Our analyses on a functional relationship between EMT and activation of glycolysis uncovered a direct link between the malignancy-driving EMT mechanism in breast cancer progression and metabolic reprogramming of the tumor cells. Specifically, we identified Twist1, a pro-metastatic master regulator of EMT as a direct modulator of a key glycolytic enzyme known to modulate the rate of glycolysis, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3). We found that Twist1 binds directly to the promoter region of PFKFB3 and induces expression of the enzyme. Additionally, inhibition of Twist1 expression in TES2b (TES2b-shTwist1) cells decreased glycolytic flux, while Twist1 overexpression in HMLE immortalized human breast epithelial cells (HMLE-Twist1) increased glycolytic activity and simultaneously decreased cellular respiration. Metabolomics analyses of HMLE vs. HMLE-Twist1 cells using Gas Chromatography Mass Spectroscopy (GCMS) indicated that Twist1 expression is sufficient to induce drastic changes in the overall metabolic state of the cells, including glutamine metabolism and availability of TCA cycle intermediates and amino acids. Together, our findings are significant as they underscore the importance and potency of EMT activation in cancer and uncover a novel mechanism by which EMT activation through Twist1 drives metabolic alterations that fuel cancer progression and metastasis. Results from this study will ultimately aid in the discovery and development of new paradigms to fight cancer mortality rates around the world. Citation Format: Esmeralda Casas, Antonio F. Santidrian, Mihaela Lorger, Ali Torkamani, Boris Ratnikov, Sheena M. Saayman, Julie B. Steele, Smith W. Jeffrey, Brunhilde Felding. Twist1 modifies the metabolic state of breast cancer cells through transcriptional activation of a key regulator of glycolysis. [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 57.

Abstract 4560: Induced Sprouty4 expression in breast invasive ductal carcinoma cells downregulates ERK/MAPK activity and restores an epithelial-like phenotype

Abstract Breast cancer is one of the most common cancers in U.S. women, with approximately 25% of these cases being in situ disease. Sprouty proteins are currently recognized as important regulators of ERK/MAPK signaling, and have been studied in various cancer types. By employing RNA-seq, previous studies from our laboratories determined specific gene expression changes common to three models of ductal carcinoma in situ (DCIS)—MCF10.DCIS, SUM 102, and SUM 225—when compared to MCF10A cells (a model of non-transformed breast epithelium). All cell lines were grown in three-dimensional (3D) reconstituted basement membrane overlay culture because research has shown that the behavior of cancer cells in 3D matrices is more reflective of an in vivo response when exposed to drugs and radiotherapy than if they are cultured on plastic. Among the three models of DCIS, 63 genes were consistently upregulated. We identified 244 promoters associated with these 63 genes and performed bioinformatic data mining that revealed a high level of enrichment for a promoter framework shared by three of these genes: one of which encoded for the protein Sprouty4. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling in DCIS, and its loss or reduced expression is a mechanism by which triple-negative lesions progress toward invasive ductal carcinoma (IDC). Using immunohistochemistry we found that Sprouty4 was highly expressed in certain human premalignant breast tissue samples, and that this expression was reduced in malignant triple-negative samples. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient over-expression of Sprouty4 reduced both ERK/MAPK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments revealed data consistent with the relocation of E-cadherin back to the cell surface and the restoration of adherens. To determine if these effects were due to changes in ERK/MAPK signaling IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug produced a restoration of an epithelial-like phenotype similar to Sprouty4 over-expression. From these data we conclude that Sprouty4 may act to control ERK/MAPK signaling in a subset of DCIS, thus limiting the progression of these premalignant breast cancers. Citation Format: Ethan J. Brock, Ryan Jackson, Seema Shah, Quanwen Li, Mansoureh Sameni, Stephen A. Krawetz, Shihong Mao, Bonnie F. Sloane, Raymond R. Mattingly. Induced Sprouty4 expression in breast invasive ductal carcinoma cells downregulates ERK/MAPK activity and restores an epithelial-like phenotype. [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 4560.

Abstract 5095: Hic-5 modulation of the stromal matrix is required for mammary tumor progression

Abstract Primary tumors grow and invade into surrounding tissues/stroma, frequently resulting in metastasis to distant organs. Tumor cell-stroma crosstalk plays a crucial, but incompletely understood role during tumor progression and metastasis. For example, increased stromal matrix density and/or rigidity correlates with poor prognosis in human breast cancer patients. Previous studies have implicated Hic-5, a focal adhesion scaffold protein, in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in mammary tumor progression, Hic-5 -/- mice were generated and crossed with mice expressing Polyoma Middle T Antigen (PyMT) driven by the MMTV promoter. The PyMT mouse is a well-established model for human breast cancer progression. Tumors from the Hic-5 -/- PyMT mice demonstrated an increased latency and had reduced growth as compared to Hic-5 +/- PyMT controls. The absence of Hic-5 did not affect the progression of the tumor to a carcinoma stage, but there was a decrease in metastasis to the lungs. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer associated fibroblasts (CAFs) and the Hic-5 -/- PyMT tumor stroma exhibits reduced extracellular matrix (ECM) deposition. Furthermore, 3D cell-derived matrices (CDM) generated in vitro using isolated CAFs, confirmed that Hic-5 -/- PyMT CAFs do not efficiently deposit and organize ECM. The organization of the stromal matrix has been shown previously to increase the rigidity of the tumor, which in turn promotes FAK Y397 phosphorylation in the tumor cells to promote cell invasion and proliferation. Accordingly, the Hic-5 -/- PyMT tumors exhibited a reduction in FAK phospho-Y397 staining and attenuation of downstream ERK activation, suggesting that their stromal matrix is not optimally organized for tumor cell signaling. Taken together, these data demonstrate that Hic-5 expression in CAFs is important for stromal matrix organization/remodeling during tumor progression and that the loss of the organization of the ECM in the Hic-5 -/- tumor stroma may contribute to the reduced tumor growth and metastasis observed in these animals. Supported by NIH CA163296 to CET, NS066071 to EO and Carol M. Baldwin Breast Cancer Fund to CET. Citation Format: Gregory J. Goreczny, Jessica Ouderkirk, Eric Olson, Mira Krendel, Christopher Turner. Hic-5 modulation of the stromal matrix is required for mammary tumor progression. [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 5095.

Suppression of endogenous lipogenesis induces reversion of the malignant phenotype and normalized differentiation in breast cancer.

The correction of specific signaling defects can reverse the oncogenic phenotype of tumor cells by acting in a dominant manner over the cancer genome. Unfortunately, there have been very few successful attempts at identifying the primary cues that could redirect malignant tissues to a normal phenotype. Here we show that suppression of the lipogenic enzyme fatty acid synthase (FASN) leads to stable reversion of the malignant phenotype and normalizes differentiation in a model of breast cancer (BC) progression. FASN knockdown dramatically reduced tumorigenicity of BC cells and restored tissue architecture, which was reminiscent of normal ductal-like structures in the mammary gland. Loss of FASN signaling was sufficient to direct tumors to a reversed phenotype that was near normal when considering the development of polarized growth-arrested acinar-like structure similar to those formed by nonmalignant breast cells in a 3D reconstituted basement membrane in vitro. This process, in vivo, resulted in a low proliferation index, mesenchymal-epithelial transition, and shut-off of the angiogenic switch in FASN-depleted BC cells orthotopically implanted into mammary fat pads. The role of FASN as a negative regulator of correct breast tissue architecture and terminal epithelial cell differentiation was dominant over the malignant phenotype of tumor cells possessing multiple cancer-driving genetic lesions as it remained stable during the course of serial in vivo passage of orthotopic tumor-derived cells. Transient knockdown of FASN suppressed hallmark structural and cytosolic/secretive proteins (vimentin, N-cadherin, fibronectin) in a model of EMT-induced cancer stem cells (CSC). Indirect pharmacological inhibition of FASN promoted a phenotypic switch from basal- to luminal-like tumorsphere architectures with reduced intrasphere heterogeneity. The fact that sole correction of exacerbated lipogenesis can stably reprogram cancer cells back to normal-like tissue architectures might open a new avenue to chronically restrain BC progression by using FASN-based differentiation therapies.

Abstract IA07: Systemic regulation of breast cancer progression

Abstract Recurrent disease affects approximately 30% of breast cancer patients but, unfortunately, by the time overt metastases are detected, it is no longer possible to treat breast cancer patients with curative intent. Clinical evidence indicates that in women who are diagnosed with metastatic breast cancer, tumor cells disseminate from the primary tumor prior to surgical resection but can remain indolent for varying periods of time before the patient becomes symptomatic. The reasons why certain disseminated cells remain indolent while others erupt into malignant tumors remain unknown; it is therefore currently difficult to predict with any degree of accuracy which patients will experience disease relapse. Our lab uses various mouse and xenograft models of breast cancer progression to study the early phases of metastatic disease when patients harbor indolent disseminated tumor cells in the periphery at the time of their primary diagnosis. Using triple-negative models, we previously showed that certain tumor systemically support the outgrowth of distant, otherwise indolent disseminated tumors, doing so by secreting cytokines into the host circulation and activating bone marrow hematopoietic cells to become pro-tumorigenic (McAllister, et al., Cell, 2008; Elkabets, et al., JCI, 2011; Castano, et al, Ca Disc, 2013). Using models of luminal breast cancer, we discovered that certain tumors promote the outgrowth of distant tumors by supporting angiogenesis (Kuznetsov, et al., Ca Disc, 2012). More recently, we have begun to identify the properties of disseminated tumor cells that are poised to respond to systemic factors and importantly, the phenotype of those that do not respond to systemic regulation. We have learned that certain systemic factors (cytokines and hematopoietic cells) elicited by primary breast tumors can sustain tumor-initiating properties of disseminated cells. Our findings highlight the systemic environment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from neoadjuvant or adjuvant therapy. Moreover, we hope that this work will pave the way to designing new therapies that can be delivered at a time when what has previously been an inexorable progression to terminal metastatic disease may be prevented. Citation Format: Sandra S. McAllister. Systemic regulation of breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA07.

FAK tyrosine 407 organized with integrin αVβ5 in Hs578Ts(i)8 advanced triple-negative breast cancer cells.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase known to promote cell migration and invasiveness. Overexpression and increased activity of FAK are closely associated with metastatic breast tumors and are linked to poor prognosis. This study discovered an inverse correlation between FAK activity and migratory and invasive behavior. We show decreased phosphorylation levels of FAK at tyrosine residues 397 and 861, and most prominently at Y407, in the more invasive Hs578Ts(i)8 subclone of the Hs578T breast cancer progression model. There is limited information available on FAKY407, and here we demonstrate its presence in triple-negative breast cancer (TNBC) cell lines. Furthermore, our studies propose that localization of FAKY407, rather than FAK expression and overall FAKY407 phosphorylation levels, is crucial for the control of cell motility. FAKY407 is found extensively at the cell periphery in focal adhesion-like structures at each end of actin stress fibers and organized with integrin αVβ5 receptors, linking the αVβ5 integrin-mediated migratory behavior of Hs578Ts(i)8 cells to FAKY407. These data suggest that subcellular localization, next to expression and activity levels, are important for understanding TNBC progression. Such an approach opens new avenues for further studies and may provide novel insight for the classification of TNBC and facilitate the discovery of effective biomarkers for diagnosis and therapy of TNBC.

Spatiotemporal progression of metastatic breast cancer: a Markov chain model highlighting the role of early metastatic sites.

Background:Cancer cell migration patterns are critical for understanding metastases and clinical evolution. Breast cancer spreads from one organ system to another via hematogenous and lymphatic routes. Although patterns of spread may superficially seem random and unpredictable, we explored the possibility that this is not the case.Aims:Develop a Markov based model of breast cancer progression that has predictive capability.Methods:On the basis of a longitudinal data set of 446 breast cancer patients, we created a Markov chain model of metastasis that describes the probabilities of metastasis occurring at a given anatomic site together with the probability of spread to additional sites. Progression is modeled as a random walk on a directed graph, where nodes represent anatomical sites where tumors can develop.Results:We quantify how survival depends on the location of the first metastatic site for different patient subcategories. In addition, we classify metastatic sites as “sponges” or “spreaders” with implications regarding anatomical pathway prediction and long-term survival. As metastatic tumors to the bone (main spreader) are most prominent, we focus in more detail on differences between groups of patients who form subsequent metastases to the lung as compared with the liver.Conclusions:We have found that spatiotemporal patterns of metastatic spread in breast cancer are neither random nor unpredictable. Furthermore, the novel concept of classifying organ sites as sponges or spreaders may motivate experiments seeking a biological basis for these phenomena and allow us to quantify the potential consequences of therapeutic targeting of sites in the oligometastatic setting and shed light on organotropic aspects of the disease.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

Abstract 2244: Functional interactions between estrogen-related-receptor β (ESRRB) and transforming growth factor-beta (TGF-β) in the regulation of breast cancer stem cell dynamics

Abstract Breast cancer is a worldwide problem that accounts for almost a quarter of all cancers in women; however, better therapeutic approaches are required since it is estimated that one to three deaths from overtreatment occur for every one death avoided. Our lab is interested in transforming growth factor beta (TGF-β) signaling and its dual role as tumor suppressor/promoter in breast cancer and its therapeutic applications. We previously performed genome-wide ChIP/Chip analysis to identify TGF-β-activated Smad3 target genes in a model of breast cancer progression. We discovered that the estrogen-related-receptor β (ESRRB) transcription factor binding motif was significantly enriched in Smad3 binding regions in breast cancer cell lines. The data suggested that functional interactions between ESRRB and the TGF-β pathway may influence breast cancer progression. ESRRs (α, β, γ) are members of the nuclear orphan receptor family that share significant homology with the estrogen receptors but are not activated by natural estrogens. Additionally, ESRRB maintains pluripotency in embryonic stem cells (ESCs) and is activated by Wnt signaling to promote self-renewal in ESCs. Thus we hypothesized that mechanistically ESRRB/TGF-β may affect breast cancer progression through effects on cancer stem cell (CSC) dynamics and cancer cell differentiation. We showed that ESRRB protein is overexpressed in human breast cancer compared with matched normal breast tissue, and that high expression of ESRRB colocalizes with the stem cell master regulator OCT4 in human breast cancer xenografts. We performed ESRRB knockdown in the MCF10Ca1h, MCF10Ca1a and MDAMB231 breast cancer cell lines and our data demonstrate that ESRRB opposes the inhibitory effects of TGF-β on CSCs as measured by tumorsphere formation assay, while having little or no effect on proliferation of the bulk tumor cell culture in vitro. More importantly, knockdown of ESRRB reduced the in vivo tumorigenicity of all three breast cancer lines and enhanced their histologic differentiation. Extreme limiting dilution assays in vivo showed that knockdown of ESRRB in MDAMB231 cells caused a 78-fold decrease in the relative number of CSCs. In conclusion, our preliminary findings suggest that ESRRB antagonizes the inhibitory effects of TGF-β on CSCs and breast cancer progression, making ESRRB an attractive therapeutic target whose inhibition can restore the tumor suppressive effects of TGF-β and reduce the tumorigenic breast CSC population. Citation Format: Nellie Moshkovich, Misako Sato, Binwu Tang, Yu-an Yang, Kathleen C. Flanders, Mitsutaka Kadota, Howard Yang, Maxwell P. Lee, Lalage M. Wakefield. Functional interactions between estrogen-related-receptor β (ESRRB) and transforming growth factor-beta (TGF-β) in the regulation of breast cancer stem cell dynamics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2244. doi:10.1158/1538-7445.AM2015-2244

Abstract P1-07-30: FOXC1/FOXA1 transcriptional balance in breast cancer: From acquisition of mesenchymal and stem cell traits to occult lymph node independent breast cancer metastasis

Abstract Background: Distant metastatic spread of cancer cells to other organs from the primary site of origin currently constitutes the most significant contributor to cancer-related morbidity and mortality. Epithelial-to-mesenchymal transition (EMT) is a biologic transformation of cancer cells from a non-migratory phenotype to a migratory one, and is thought to initiate the metastatic cascade in cancer. EMT has also been reported to trigger acquisition of stem cell traits in breast cancer. Transcription factor (TF) Forkhead box C1 (FOXC1), strongly associated with the basal-like and claudin-low breast cancer molecular subtypes, is a powerful EMT inducer and is also a marker of stem/progenitor cells. In contrast, TF forkhead box A1 (FOXA1), strongly associated with luminal subtypes, is an EMT repressor and a luminal differentiation marker, thus seemingly exerting reciprocally opposite transcriptional effects to that of FOXC1. We hypothesized that effective EMT program activation status in breast cancer might be better predicted by examining the expression ratio of an EMT inducer and EMT repressor, such as FOXC1/FOXA1, theoretically being more reflective of net transcriptional effect than either component alone. Methods: Herein we utilize RNA-Seq profiling of the HRAS-transformed MCF10A cell series, a well characterized and widely accepted in vitro model of breast cancer progression and metastasis, to correlate measured FOXC1/FOXA1 ratios to dynamic shifts in EMT marker expression in 3D matrigel cultures and to stem cell traits observed in primary and secondary mammosphere suspension cultures. We further test the ability of the FOXC1/FOXA1 expression ratio to predict lymph node independent breast cancer metastasis and death in independent human breast cancer gene expression datasets. Results: RNA-Seq and qRT-PCR profiling confirmed progressive increase in FOXC1/FOXA1 ratio to correlate with a progressive loss of E-cadherin expression and synchronous gain of EMT markers N-cadherin, Fibronectin, and Vimentin. FOXC1/FOXA1 ratio was found to be directly proportional to mammosphere formation efficiency, a surrogate indicator of stem cell enrichment. In patients without any evidence of nodal metastasis, elevated FOXC1/FOXA1 ratio was associated with significantly decreased 10 year Overall Survival (HR 2.58;95%CI 1.39 to 4.80, p = 0.003, 295 patient Van de Vijver dataset), 10 year Disease-specific Survival (HR 1.74;95%CI 1.16 to 2.61, p = 0.008,1992 patient Curtis dataset) and predicted the development of lung metastasis. Conclusion: Elevated FOXC1/FOXA1 expression ratio indicates EMT program activation in breast cancer, and predicts the associated occurrence of lymph-node-independent distant metastasis and death in human patients. These findings may allow for the early (pre-symptomatic) diagnosis of clinically occult (node negative) metastasis by using the FOXC1/FOXA1 ratio as a biomarker of metastasis and permit institution of appropriate therapy earlier than currently possible. The current study improves our understanding of EMT and highlights the importance of future studies geared towards unraveling mechanisms involved in regulating FOXC1 and FOXA1 expression in breast cancer. Citation Format: Partha S Ray, Tor W Jensen, Tania Ray, Omid Gholamalamdari, Connie Y Tsai, Bomy Kim, Rohit Bhargava, K V Prasanth. FOXC1/FOXA1 transcriptional balance in breast cancer: From acquisition of mesenchymal and stem cell traits to occult lymph node independent breast cancer metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-30.

Abstract P3-04-16: Estrogen receptor β agonists reduce breast cancer tumor growth in syngeneic mouse models

Abstract The estrogen receptors (ER) play a significant role in breast cancer, with the majority of breast cancers expressing estrogen receptor alpha (ERα) and depending on its signaling. ERα has proliferative function, however, estrogen receptor beta (ERβ) has anti-proliferative functions. Recently, several selective ERβ agonists have been identified. The objective of this study was to determine the effectiveness of ERβ agonists in inhibiting the growth of distinct breast cancer cells both in-vitro and in-vivo, and to determine the mechanisms involved. The mouse mammary tumor cell lines, D2A1 and MM51, express both ERα and ERβ. Specifically D2A1 cells are dependent on estrogen for growth and have increased expression of aromatase. Additionally, D2A1 cells are very aggressive and highly metastatic; representing a good model of breast cancer progression. MM51 cells are Her2/neu positive and are an adequate model to study the crosstalk between growth factors and ER signaling. Syngeneic mouse tumor models were used to incorporate an intact immune system which plays a role in the tumor’s microenvironment. Our in-vitro studies demonstrate that ERβ agonists significantly inhibit cell growth in a dose dependent manner. Our syngeneic studies show that, in-vivo, ERβ agonists effectively reduce tumor volume and inhibit tumor progression. This study reveals that in addition to acting on ERβ, these agonists reduce the expression of ERα at both the mRNA and protein level, therefore modulating the ratio of ERα to ERβ. Additionally, treatment with ERβ agonists results in increases apoptosis through increased p53 expression, and cell cycle arrest through p27 and cyclin D1. Together, these studies demonstrate the therapeutic potential of ERβ agonists for the treatment of breast cancer. Citation Format: Cathy Samayoa, Naveen K Krishnegowda, Ratna K Vadlamudi, Rajeshwar R Tekmal. Estrogen receptor β agonists reduce breast cancer tumor growth in syngeneic mouse models [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-16.

Abstract P6-07-06: Post-transcriptional coordination of gene expression during breast cancer tumorigenesis

Abstract We investigate mechanisms of RNA regulation central to human breast cancer progression. Aberrant gene expression is known to be an important factor in cancer, yet little is known about the role RNA-binding proteins (RBPs) play in disease onset and progression. Sequence specific RBPs coordinately regulate subsets of functionally related mRNAs within ribonucleoprotein complexes (RNPs), which are remodeled in response to cellular perturbations, allowing for the rapid and coordinated translation of proteins that have common functions. These post-transcriptional events robustly influence expression patterns of proto-oncogenes, growth factors, cytokines, and cell cycle regulators by influencing both mRNA stability and translation. Therefore, understanding the post-transcriptional layer of gene regulation is critical to understanding cancer development and progression. We have identified significant transcriptomic changes during the stepwise transition from primary mammary epithelial cells to a fully malignant state, as well as coordinated RNA dynamics of transient cellular RNP complexes. For this analysis, we generated a model of human breast cancer formation in which normal mammary epithelial cells were first immortalized through the expression of hTERT, p53DD, Cyclin D1, CDK4 R24C and C-MYCT58A, and then subsequently transformed by the addition of H-RASG12V. RNA-sequencing analysis demonstrated that the genes most significantly changed in this model are those involved in cell adhesion. In fact, while primary cells have a gene expression pattern typical of normal epithelial cells, both immortalized and transformed cells exhibit an mRNA expression pattern typical of mesenchymal cells. Interestingly, we found that N-cadherin protein, as well as other prototypical mesenchymal proteins, are only robustly expressed in the fully malignant cells, but not in immortalized cells. This suggests coordinated translational regulation of mRNAs that are essential for activating the epithelial-to-mesenchymal transition (EMT). Many of these EMT-related mRNAs are targets of both the HuR protein and microRNAs. HuR is a translational activator that competes with microRNAs and is mislocalized in many cancers. To integrate the quantitative RNA dynamics of HuR and microRNAs on a global scale, we used RNP-Immunoprecipitation followed by high-throughput sequencing to identify and quantify the remodeling of mRNA subsets associated with HuR and the Ago2/RISC complex in our breast cancer progression model. This integrative work provides global information about the underlying biology of carcinogenesis at the level of post-transcriptional coordination of gene expression, resulting in a more comprehensive understanding of the many layers of complex gene regulation. Therefore, the results from this study may ultimately direct our ability to counter the RNA regulatory changes that underlie malignancy. Citation Format: Laura Simone Bisogno, Jack D Keene. Post-transcriptional coordination of gene expression during breast cancer tumorigenesis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-06.

Cost-effectiveness of breast cancer screening policies using simulation

In this paper, we study breast cancer screening policies using computer simulation. We developed a multi-state Markov model for breast cancer progression, considering both the screening and treatment stages of breast cancer. The parameters of our model were estimated through data from the Canadian National Breast Cancer Screening Study as well as data in the relevant literature. Using computer simulation, we evaluated various screening policies to study the impact of mammography screening for age-based subpopulations in Canada. We also performed sensitivity analysis to examine the impact of certain parameters on number of deaths and total costs. The analysis comparing screening policies reveals that a policy in which women belonging to the 40–49 age group are not screened, whereas those belonging to the 50–59 and 60–69 age groups are screened once every 5 years, outperforms others with respect to cost per life saved. Our analysis also indicates that increasing the screening frequencies for the 50–59 and 60–69 age groups decrease mortality, and that the average number of deaths generally decreases with an increase in screening frequency. We found that screening annually for all age groups is associated with the highest costs per life saved. Our analysis thus reveals that cost per life saved increases with an increase in screening frequency.

Mechanobiology of mRNA Localization in Breast Cancer Cells

Introduction: Metastasis occurs when cancer cells form secondary tumors in distant areas of the body. Localization of RNAs in lamellipodial regions has been proposed to play an important role during metastatic progression. In one pathway, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cell protrusions. APC -associated mRNAs have been implicated in cellular migration and metastatic progression. Therefore, localization or not of these mRNAs has functional significance in cellular migration and metastasis. Additionally, It has been shown that cancer cells modulate their gene expression in response to the mechanical properties of the substrate. Furthermore, mRNA localization at adhesion sites is influenced by mechanical tension, which is adjusted by cells as a function of the mechanical properties of the cell environment. Therefore, mechanical properties of tissues may play a role during metastasis by modulating localization of mRNAs. As a result, this study investigates APC- associated mRNA localization as a function of substrate stiffness. Methods: We used the MCF10A cell series, a breast cancer progression model composed of cell lines representing pre-malignant to invasive transformation to investigate mRNA localization. By using in situ hybridization to fluorescently label mRNAs, and micropatterned polyacrylamide gels of varying stiffness, we observed APC associated mRNA localization. Glu-tubulin and vinculin were immunostained to study their relationship to mRNA localization. Results: On stiffer substrates (280kPa), we observed increased mRNA localization compared to softer substrates (0.87kPa). Staining of cytoskeletal elements such as Glu-tubulin and vinculin showed a correlation between the abundance and location of the proteins and APC-associated mRNAs. These results reveal biophysical roles of adhesion and cytoskeletal tension in regulating mRNAs to protrusions.

Abstract A46: Breast cancers come out of nowhere: A two part Durham Community/Duke partnership to identify signaling pathways during the initiation of triple-negative breast cancer in African American women

Abstract Background: The current trend of increased survivability of women with breast cancer has been linked to increased awareness, early detection and better treatment. In fact the American Cancer Society estimate that there are more than 2.5 million breast cancer survivors living in America today. However the trends of these overall numbers are expressed quiet differently in the African American community. African American women have a 77% five-year survival rate while survival rates for European American are at 90%. It is clear that lack of access plays a role in poor survival of African American women. Other factors are hypothesized to be aggressive biology and lack of early detection strategies for triple-negative breast cancer. Methods and Results: Phase I: We developed a community outreach partnership with the African American community in Durham, NC. One of the concerns of the community was that aggressive breast cancers “appeared to come out of nowhere” and seemed to defy the conventional model of stepwise breast cancer progression. This alliance allowed the development of a partnership to use breast Magnetic Resonance Imaging (MRI) screening to evaluate the onset of breast cancer occurring in over 300 African American high-risk women (>48 month observation, 12 cancers). The results of this study corroborated the concerns of the community, that incidence of breast cancer could indeed occur in less than 6 months in women who had previously had a normal MRI. The results suggested that these aggressive breast cancers displayed an accelerated initiation and development within the patients of the local African American community, validating community observations. Phase II: The second concern raised by our community partners was the lack of prevention agents for these aggressive breast cancers. In partnership with Durham community members, our current study investigated signaling pathways activated during the initiation of triple-negative breast cancer in African American women. For our study 10 African American women who were at highest risk for breast cancer (either known or suspected BRCA1 mutation carriers) donated mastectomy tissue. Women either underwent prophylactic mastectomy or had mastectomy after a cancer diagnosis. Donated mastectomy specimens were serial sliced into 2.5 centimeter slices and 2 to 3 cubic centimeter samples were excised from specified section of each slice of breast tissue. Breast tissues were snap frozen, formalin-fixed and OCT embedded to preserve samples for further analysis. Thus far tissue tensile forces, collagen composition, metabolic and proteomic profiles have been characterized for this cohort. In addition biomarkers for stem cell, proliferation, apoptosis and senescence have been established. Conclusion: Our Durham Community/Duke Partnership has allowed us to study a unique phenotype displayed in pre- malignant breast lesion found in rapid onset breast cancers displayed in high-risk African American women. Our study highlights the power of investigators working in collaboration and partnership with the community. Citation Format: Christopher Sistrunk, Stephaine Robertson, Adrian Ambrose, Catherine Ibarra Drendall, Victoria Seewaldt. Breast cancers come out of nowhere: A two part Durham Community/Duke partnership to identify signaling pathways during the initiation of triple-negative breast cancer in African American women. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr A46. doi:10.1158/1538-7755.DISP13-A46

Abstract 2109: All trans-retinoic acid (ATRA) induces redifferentiation of early transformed breast epithelial cells

Abstract Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic, and anti-oxidant properties. The effects of retinoids are mediated by retinoic acid receptors (RARβ;,RARα,RARγ) and the retinoid X receptors (RXR) that bind the DNA as heterodimers. The objective of this project was to study the potential of retinoic acid to revert the neoplastic process using an in vitro model of breast cancer progression. This model was previously developed by treating the human normal breast epithelial MCF-10F cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: (a) the spontaneously immortalized cell line MCF-10F, which does not show any characteristic of invasiveness or tumor formation and therefore is considered to be a normal-like breast epithelial cell line; (b) the transformed trMCF cells; (c) the invasive bsMCF cells and; (d) the caMCF tumor cells. The bsMCF cells induced tumors when they were injected in the mammary fat pad of SCID mice. We previously showed that RARβ was unmethylated in MCF-10F and trMCF cells, but became hypermethylated in bsMCF and caMCF. We studied the effect of retinoic acid at the different stages in this model by cultivating the cells in 3D-cultures (collagen matrix). In collagen, the MCF-10F cells form tubules resembling the normal mammary gland. After treatment with estradiol, cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with different concentrations of ATRA (10-5M to 10-8M). We found that trMCF clone 11 cells showed a reduction of solid masses and increased number of tubules in collagen after being treated with 10-6M ATRA. A total of 43% of the structures were tubules in trMCF clone 11 cells treated with 10-6M ATRA. Gene expression studies showed that 207 genes up-regulated in transformed trMCF clone 11 cells were down-regulated after ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were down-regulated in trMCF clone 11 were up-regulated after ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 10-6M ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process to a normal stage and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after treatment with ATRA alone or in combination with 5-aza-dC. These results suggest that ATRA could be used as chemopreventive agent to inhibit the progression of premalignant lesions of the breast. This work was supported by The Pennsylvania Breast Cancer Coalition and Friends for an Earlier Breast Cancer Test. Citation Format: Maria F. Arisi, Rebecca A. Starker, Sankar Addya, Yong Huang, Sandra V. Fernandez. All trans-retinoic acid (ATRA) induces redifferentiation of early transformed breast epithelial 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 2109. doi:10.1158/1538-7445.AM2014-2109

Abstract 3388: The CD44s splice isoform is essential for breast tumor initiating cell properties

Abstract Tumor initiating cells (TICs) constitute a small fraction of tumor cells that display stem cell characteristics, including the ability to self-renew and generate heterogeneous tumors. A CD44+/CD24- profile has been widely used to identify TICs in a variety of tumors. However, it has remained unknown whether CD44 is functionally required for TICs and whether a specific CD44 isoform is enriched in TICs. Here, we report that the CD44s isoform is the major splice isoform expressed in a CD44+/CD24- breast stem cell population. Functionally, CD44s is required for expression of a breast stem cell (BSC) gene signature and is essential for mammosphere formation. Modulation of CD44 alternative splicing by the splicing regulator ESRP1 prevents CD44s production and, in turn, inhibits mammosphere formation and expression of the BSC signature. Furthermore, CD44s is required for limiting numbers of TICs to form tumors in an animal model of breast cancer progression. Together, these data illustrate that the CD44s splice isoform is enriched in CD44+/CD24- breast stem cells and serves as an important regulator of stem cell properties. Our results also suggest that CD44s-dependent TIC properties can be controlled at the level of alternative splicing by splicing factors that regulate production of specific CD44 isoforms. Citation Format: Rhonda Brown, Sali Liu, Lauren Reinke, Chonghui Cheng. The CD44s splice isoform is essential for breast tumor initiating cell properties. [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 3388. doi:10.1158/1538-7445.AM2014-3388