Joeli Brinkman

ER Re-expression and Re-sensitization to Endocrine Therapies in ER-negative Breast Cancers

Breast cancer is the leading cause of cancer amongst women in the westernized world. The presence or absence of ERα in breast cancers is an important prognostic indicator. About 30–40% of breast cancers lack detectable ERα protein. ERα− breast cancers are resistant to endocrine therapies and have a worse prognosis than ERα+ breast cancers. Since expression of ERα is necessary for response to endocrine therapies, investigational studies are ongoing in order to understand the generation of the ERα− phenotype and develop interventions to restore ERα expression in ERα− breast cancers. DNA methylation and chromatin remodeling are two epigenetic mechanisms that have been linked with the lack of ERα expression and in these cases; demethylation of the ERα promoter or treatment with HDAC inhibitors shows promise in restoring ERα expression in ERα− breast cancers. Two additional potential mechanisms underlying generation of the ERα− phenotype involve E6-AP and Src, both of which have been shown to be elevated in ERα− breast cancer and can drive the proteasomal degradation of ERα. Recently, studies have demonstrated that upregulated growth factor signaling due to hyperactive MAPK activity significantly contributes to generation of the ERα− phenotype and that inhibition of MAPK activity can cause re-expression of the ERα and restore sensitivity to endocrine therapies. Given the challenges in treating ERα− breast cancer, understanding and manipulating the cellular mechanisms that effect expression of ERα are imperative in order to restore sensitivity to endocrine therapies and to design novel therapeutics for the treatment of ERα− breast cancers.

A Novel MAPK–microRNA Signature Is Predictive of Hormone-Therapy Resistance and Poor Outcome in ER-Positive Breast Cancer

Purpose: Hyperactivation of ERK1/2 MAPK (hMAPK) leads to loss of estrogen receptor (ER) expression and poor outcome in breast cancer. microRNAs (miRNA) play important regulatory roles and serve as biomarkers of disease. Here, we describe molecular, pathologic, and clinical outcome associations of an hMAPK–miRNA expression signature in breast cancer. Experimental Design: An hMAPK–miRNA signature was identified, and associations of this signature with molecular and genetic alterations, gene expression, pathologic features, and clinical outcomes were determined in primary breast cancers from training data and validated using independent datasets. Univariate and multivariate analyses identified subsignatures associated with increased disease recurrence and poorer disease survival among ER-positive (ER+) patients, respectively. Results: High-hMAPK–miRNA status significantly correlated with ER-negativity, enrichment for basal and HER2-subtypes, and reduced recurrence-free and disease-specific survival in publicly available datasets. A robust determination of a recurrence signature and a survival signature identified hMAPK–miRNAs commonly associated with poor clinical outcome, and specific subsets associated more closely with either disease recurrence or disease survival, especially among ER+ cancers of both luminal A and luminal B subtypes. Multivariate analyses indicated that these recurrence and survival signatures significantly associated with increased risk of disease-specific death and disease recurrence in ER+ cancer and ER+ cancers treated with hormone therapy. Conclusions: We report an hMAPK–miRNA signature and two subsignatures derived from it that associate significantly with adverse clinical features, poor clinical outcome, and poor response to hormone therapy in breast cancer, thus identifying potential effectors of MAPK signaling, and novel predictive and prognostic biomarkers or therapeutic targets in breast cancer. Clin Cancer Res; 21(2); 373–85. ©2014 AACR.

Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures

Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as “dissociated tumor (DT) cells.” In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, including PAM50 predictor analysis. In vivo, tumorigenic and metastatic potential of DT cultures was assessed in NOD/SCID and NSG mice. These cellular models differ from recently developed patient-derived xenograft models in that they can be used for both in vitro and in vivo studies. PAM50 predictor analysis showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. Three DT cultures comprised of cancer-associated fibroblasts (CAFs) were isolated from luminal A, Her2-enriched, and basal primary tumors. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, offering novel cellular models of ER-negative breast cancer subtypes. A group of CAFs provide tumor subtype-specific components of the tumor microenvironment (TME). Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis, and TME.

Abstract B108: Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs

The tumor microenvironment (TME) has been shown to play a vital role in tumor development and progression. Carcinoma associated fibroblasts (CAFs) form a very essential component of the TME. In fact, the TME in different types of carcinomas, particularly carcinomas of the breast, is comprised mainly of fibroblasts. CAFs in breast cancer TME secrete cytokines and growth factors that are known to activate a multitude of signaling pathways in breast cancer cells. One of these pathways is the MAPK pathway, a signaling pathway activated downstream of ERBB receptor tyrosine kinase family members. We have previously demonstrated that activation of the MAPK pathway represses estrogen receptor (ER) expression leading to an ER-negative phenotype in breast cancer. We have generated dissociated CAF cell cultures from ER+, ER-/Her2+, and triple negative (TN) primary breast tumors. We have characterized these CAFs on the basis of fibroblast markers, epithelial markers, microarray analysis, soft agar assay and in vivo tumorigenecity studies. Our results show that these CAF populations from primary breast tumors are pure populations of CAF cell cultures. These CAFs provide a model that allows us to examine the role of CAF interaction with ER+ breast cancer cells regarding activation of MAPK and subsequent repression of ER expression. We demonstrate that treatment of the ER+ MCF-7 cell line with conditioned media (CM) from the CAFs results in transient activation of MAPK signaling and subsequent repression of ER. Continuous exposure of ER+ breast cancer cells to soluble factors from CAFs also results in activation of MAPK and down-regulation of ER expression. Importantly, CM from human mammary fibroblasts (HMFs) and CAFs generated from an ER+ tumor do not down-regulate ER expression. Gene expression analysis and cytokine arrays indicate key differences in gene and cytokine expression between HMFs and the ER+ tumor CAFs compared to the two CAFs generated from ER- tumors. We have previously identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK). This signature identifies the majority of ER- breast cancers as well as a population of ER+ breast cancers that express lower levels of ER. Like the ER- breast cancers identified by this signature, ER+ cancers identified by this signature exhibit gene expression patterns indicative of activated MAPK signaling, and exhibit significantly increased disease recurrence and significantly reduced disease survival. Here we show that breast tumors cancers bearing this hMAPK-miRNA signature have elevated expression of numerous stromal markers associated with poor clinical outcome, as well as a number of microRNAs that are differentially expressed between normal human mammary fibroblasts (HMFs) and mammary tumor carcinoma CAFs. Some of these miRNAs have been established as targeting ER. Using reporter constructs to investigate microRNA regulation of ER, we have observed that treatment of ER+ breast cancer cells with CM from ER- CAFs results in enhanced microRNA-dependent repression of ER. These data are suggestive of a role for CAF secreted factors in activating MAPK in breast cancer cells leading to repression of ER. In addition, CAFs are able to increase the expression of hMAPK-miRNAs that can down-regulate ER expression either via the activation of MAPK in the cancer cell or secreted directly from the CAFs. We hypothesize that CAFs may contribute to an ER-negative phenotype in breast cancer cells not only by activating MAPK signaling in the tumor cells via secretion of growth factors, but also potentially by transfer of MAPK-regulated miRNAs to the breast cancer cells as well. Citation Format: Sanket H. Shah, Philip Miller, Katherine Drews-Elger, Joeli Brinkman, Stefania Lairet, Alana Steinberg, Dorraya El-Ashry. Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B108.

Abstract P1-05-12: Metastatic xenograft models of human estrogen receptor negative breast cancer primary cultures are driven by the recruitment of myeloid-derived suppressor cells

The study of early stage and metastatic breast cancer relies heavily on the use of established cell lines often derived from metastatic lesions rather than from primary tumors, limiting our understanding of primary tumor development. Study of the mechanisms governing the metastatic process in its entirety is not always feasible with these types of cellular and xenograft models, as often the primary tumor needs to be excised, or breast cancer cells are introduced into circulation of host mice. While this approach has allowed a better understanding of metastasis, it may unintentionally enhance the colonization efficiency and pass over important events occurring in the tumor microenvironment. In order to provide a cellular model that more closely recapitulates breast cancer development and progression, we generated breast cancer cellular cultures by dissociating specimens of human estrogen receptor-negative primary breast tumors and placing them in culture. Within our group of dissociated tumor (DT) cell cultures, we had shown that five cultures are formed by breast cancer cells and these were classified by PAM50 predictor analysis as belonging to the ER−/PR−/Her2+ and triple negative subtypes. DT cells are tumorigenic in NOD/Scid and NOD/Scid gamma (NSG) mice. Our first aim was to assess the metastatic potential of the DT cells in two mouse models. We show that 3 of the 5 cultures are metastatic to lymph nodes, liver and or lungs; common sites of metastasis in breast cancer patients. Spontaneous metastases were observed without the requirement of primary tumor excision. The metastatic frequency ranged from 25–90% in NOD/Scid mice, and from 80–100% in NSG mice. Moreover, expression of key proteins such as epidermal growth factor receptor (EGFR) detected in primary tumors was also found in the metastatic lesions. Because the DT cells have been isolated from primary breast tumors they represent a clinically relevant and valuable model to study breast cancer and metastasis. A critical factor influencing tumor progression and metastasis is the infiltration of immune cells including myeloid-derived suppressor cells (MDSCs), which have been shown to be increased in a tumor-dependent manner in xenograft models of breast cancer. Previous work from our lab has shown that stroma of tumor-bearing mice exhibited gene expression changes, including increased expression of calcium binding proteins S100A8 and S100A9, that are consistent with myeloid immune cell infiltration. Our second aim was thus to analyze the recruitment of S100A8+ MDSCs by xenografted DT tumors. We show that the recruitment of S100A8+ MDSCs is detected in DT tumors and sites of metastasis, and more importantly, their presence was significantly greater in the tumors that metastasized compared to those that did not. These data strongly suggest that the metastatic potential of breast cancer cells may be driven at least in part by the recruitment of S100A8+ MDSCs. Taken together, our observations and our closer-to-primary xenograft models may lead to a better understanding of metastatic disease, as well as to the development and pre-clinical screening of targeted breast cancer therapeutics, particularly in the metastatic setting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-12.

Abstract 3946: Investigation of a hyperactive MAPK microRNA expression profile reveals potential role for microRNAs in the generation and maintenance of ER- phenotype in breast cancer

microRNAs (miRNAs) are small RNAs around 22 nucleotides in length that function as important regulators of cell and tissue specific gene regulation, and have been shown to have critical functions in development and differentiation. Dysregulation of miRNA biogenesis and biological activity has emerged as a facet of normal biology that is usurped by cancers to promote aberrant growth and tumorigenesis. miRNA expression has been shown to discriminate among various types of cancer; for example, miRNA expression profiling correctly classifies canonical breast cancer subtypes. We have previously demonstrated that many breast cancers have enhanced signaling through the ErbB family, resulting in a gene expression signature driven by hyperactive MAPK (hMAPK) signaling that is indicative of ER status, and correlates with poor clinical outcome in breast cancer gene expression datasets. We have shown that inhibiting hMAPK signaling in a subset of ER- breast cancers results in re-expression of ER and re-establishment of anti-estrogen sensitivity. Our model system is ideally suited to study the contribution of miRNAs to the generation and maintenance of the ER- phenotype established by hMAPK signaling in breast cancer. In this study, we generated a hMAPK miRNA signature, at both the miRNA expression and miRNA activity levels. These miRNA expression and activity profiles correlate well with the hMAPK regulated mRNAs in our model of hMAPK signaling. Importantly, several of these hMAPK regulated miRNAs are known to control expression of key genes involved in the biology of ER- breast cancer, such as ER, p27KIP1, and known tumor suppressors. Pathway analysis of these miRNAs reveals potential regulation of many important biological pathways; indeed, several of these miRNAs have established roles in regulating numerous biological pathways which are often dysregulated in breast cancer. We examined the representation of this miRNA expression signature in clinical breast cancer specimens, as well as in primary cultures derived from dissociated ER- breast tumors, and investigated the correlation of this signature with various clinical features. The hMAPK miRNA profile correlates with grade, ER status, and clinical outcome. These data suggest that MAPK regulation of key miRNAs, and thus key biological pathways, is involved in establishing and maintaining phenotypes associated with hMAPK signaling in breast cancer. While mRNA and miRNA expression data independently are proven tools for tumor diagnosis and prognosis, combined analysis of gene expression and miRNA expression will provide a more powerful means for investigating and understanding the complexities of tumor biology. Here we have used such an approach to further understand and appreciate the involvement of hyperactivation of MAPK signaling pathways in the biology of ER- breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3946. doi:10.1158/1538-7445.AM2011-3946

P2-01-22: Characterization of Four ER-Negative Breast Primary Tumor Dissociated Cultures as a Model for the In Vitro and In Vivo Study of Tumorigenesis, Metastasis and Angiogenesis.

ER negative breast cancers comprise a subtype of tumors for which there are few therapies with successful response. Currently, the use of established cell lines has allowed detailed molecular and cellular studies on cancer mechanisms ranging form stem cells to proliferation, from tumorigenesis to invasion and metastases, and from tumor suppressors to oncogenes. But while cell lines provide a source of homogeneous, propagatable material, a drawback of this model is that most breast cancer cell lines are derived from metastatic lesions that may harbor additional mutations and have little resemblance with the primary tumor of origin. Additionally, the continuous passage of these cells over the last 20-plus years has given rise to well-established differences in growth rate, hormone receptor status, karyotype and clonogenicity. Our purpose here was to characterize four primary dissociated tumor (DT) cell cultures from ER- breast tumors, the establishment of which we had previously described, to determine if they could provide more accurate models for both in vitro and in vivo studies. These four DT cultures (DT16, DT22, DT25 and DT28) display a triple negative phenotype and were characterized by their genomic profile by the PAM50 predictor analysis as belonging to the basal (DT22, DT25 and DT28) and luminal B (DT16) subtypes. Of those cultures for which primary tumor sample was available, microarray gene expression profiling and cell line clustering analysis show the primary tumor and dissociated culture cluster together and to similar breast tumors. Interestingly, of the three basal DT cultures, DT22 clusters with Claudin-low tumors and cell lines. We have established that each of these 4 DTs are tumorigenic in the NOD/SCID mouse model, and are now assessing their metastatic potential. DT16 generated metastases in 25% of the mice (6 out of 24; two in liver, four in lymph node). DT22 generated highly vascularized tumors, which may provide a relevant model for study of the in vivo vascular recruitment process. In addition, we have analyzed and characterized the role of pivotal signaling pathways often deregulated in breast cancer such as EGFR, P-MAPK, PI3k/mTOR, p53, TGFb, Hedgehog, Notch, and Wnt in both the 2D culture as well as in sections of xenograft samples. Given that the scope of primary tumor heterogeneity and consequently the early stages of breast tumorigenesis are under-represented by current in vitro models in breast cancer research, the use of these DT cell cultures could more accurately recapitulate the disease in both the in vitro and in vivo settings. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-22.

P3-03-07: A Novel Approach Integrating microRNA and mRNA Signatures of HMAPK Signaling Is Highly Predictive of ER- Status and Outcome in Breast Cancer – Role of HMAPK microRNAs in Repression of ER and p27.

Deregulation of the MAPK signaling pathway in breast cancer is known to facilitate the down-regulation of the estrogen receptor and to contribute to the aggressive nature of ER negative and triple negative breast cancers. We have identified a microRNA signature indicative of hyperactive MAPK (hMAPK) signaling, which complements a previously established hyperactive MAPK gene expression signature. We have shown that hMAPK signaling also alters the regulatory activity of many microRNAs, including particular microRNAs with established roles in the biology of breast cancer, miR-221/222 and miR-22. Expression correlation with both the hMAPK microRNA signature and hMAPK mRNA signature is significantly associated with ER-negative status, increased tumor grade, high proliferation rate, and, importantly, poor disease specific survival among breast cancer patients, regardless of ER status. The hMAPK microRNA signature contains 127 microRNAs, 47 up-regulated and 70 down-regulated. Of note, hMAPK up-regulated microRNAs include miR-221/222 and 22, both of which have been demonstrated to target ER while miR-221/222 targets the cell cycle regulatory protein p27. Down-regulated microRNAs include miR-375, which positively regulates ER expression by down-regulating expression of an ER repressor. miR-221/222 and miR-22 exhibit both enhanced expression and enhanced regulatory activity in the context of hMAPK signaling, indicating an important role for these microRNAs in the biology of hMAPK signaling in breast cancer. In addition to these microRNAs, an unbiased approach of determining MAPK regulated microRNAs targeting the 3’ UTRs of both ER and p27 will identify novel microRNAs involved in the MAPK regulated repression of ER and p27. These data not only suggest a regulatory role for microRNAs whose expression and biological activity are altered under conditions of hyperactivation of MAPK signaling in establishing and maintaining ER negativity and tumor aggression, but also indicate that hMAPK signaling may represent a novel aggressive tumor biology that is indicative of poor disease outcome in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-07.

Abstract 2392: Primary cultures established from estrogen receptor negative breast tumors: Method and characterization

Approximately 35% of breast cancers lack expression of estrogen receptor (ER) protein. ER-negative (ER-) breast cancer carries a worse prognosis than ER positive (ER+) breast cancer, but more importantly, it precludes use of less toxic endocrine therapies. Our objective was to establish primary cultures from dissociation of ER- breast tumors in order to provide an alternative cellular model that can be used as a valuable tool for both in vitro and in vivo studies of ER- breast cancer. A total of eight ER- tumors were successfully dissociated into primary cultures: four from ER-/PR-/H2N- and four from ER-/PR-/H2N+ primary breast tumors with a 100% success rate. The epithelial-enriched cell pellet was placed in culture and for experiments carried for a maximum of 30 passages. These cells will be hereafter referred to as “dissociated tumor” (DT) cultures. Cells were grown in 2D culture and their in vitro morphology, proliferation rates, mammosphere and soft agar colony formation ability, and CD44/CD24 surface marker expression (tumor initiating cell (TIC) content) are determined. In addition we have performed gene expression profiling and established their tumorigenic potential in NOD/SCID female mice. Each culture exhibited its own relatively unique morphology; two of the eight cultures grew mainly as suspensions while the remaining six DT cultures grew with a higher percentage of attached, mesenchymal phenotype cells. Proliferation rates ranged from 38 to 60 hours, and while all of the DT cultures had the ability to form mammospheres, five of the eight DTs (DT13, DT16, DT22, DT25 and DT28) formed colonies in soft agar. Analysis of CD44 and CD24 surface markers expression showed that while all DT cultures were CD44 positive, expression of CD24 varied among DTs. Of the eight DT cultures, we found five of them to have a high (>85%) CD44+/CD24-/lo cell content, one the CD44+/CD24high phenotype and two of eight have populations with increasing levels of CD24 expression ranging from -/lo to medium. This CD44+/CD24-/lo phenotype is stable when examined over several passages. Microarray analysis comparing the DT cultures to cancer cell lines showed that they clustered with each other and with several breast cancer cell lines of known ER- status and EGFR or Her2 overexpression status. In agreement with the soft agar assay, DT16, DT22, DT25 and DT28 had the ability to form tumors when injected into the mammary fat pad of female NOD/SCID mice. In summary, our study describes primary cultures of dissociated ER negative breast cancer cells that provide an alternative, primary cell based model that allows both in vitro as well as in vivo experimental approaches. The use of these types of cellular models may lead to a better understanding of ER- breast cancer biology as well as being a valuable tool for screening potential therapeutic options for ER- breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2392. doi:10.1158/1538-7445.AM2011-2392

Abstract 4597: MAPK-mediated gene expression in ER-negative breast cancer: Role of miRNAs and Ago2

Background: We have previously shown that the ERα-negative phenotype in breast cancer can be driven by hyperactive MAPK signaling resulting from up-regulation of ErbB family members. We generated a hyperactive MAPK gene signature that is indicative of ERα negativity, contains many genes related to invasion, motility, metastasis, and EMT, and that shows inverse regulation of many genes known to be estrogen dependent. Recent studies have identified differential micro-RNA (miRNA) expression and activity between ERα+ and ERα- breast tumors, implying that miRNAs and their protein targets may play critical roles in both the development and maintenance of these breast cancers. Argonaute-2 (Ago2, EIF2C2), a member of the Argonaute family of proteins, associates with other proteins and miRNAs to form the RNA Induced Silencing Complex (RISC). It is also known that MAPK signaling enhances the expression and stability of Ago2, and in the absence of MAPK signaling Ago2 levels are depleted. Objective: In this study, we sought to confirm the role of MAPK signaling in Ago2 expression and stability, to determine the role of miRNAs in MAPK mediated down-regulation of ERα expression, to identify MAPK-regulated miRNAs and miRNAs whose activity is mediated through Ago2, and to investigate the roles of these miRNAs in the expression of genes in the hyperactive MAPK gene signature. Results: Inhibiting MAPK signaling in our hyperactive MAPK cell lines using the MEK inhibitor U0126 resulted in significantly decreased Ago2 expression. Conversely, hyperactivation of MAPK signaling significantly increased Ago2 expression. Using an ERα, 3′UTR-luc reporter containing the sequence known to be targeted by several miRNAs upregulated in ERα- vs ERα+ breast cancer, we determined that hyperactive MAPK drives miRNA targeting of ERα. We then generated an miRNA profile associated with hyperactive MAPK. Notably among the miRNAs whose expression is increased by MAPK are miRNA 221/222, which have been previously shown to be upregulated in ERα- breast cancer and target ERα directly. Using our MAPK gene signature, we determined the miRNAs predicted to regulate those genes, and the activities of these miRNAs in the presence and absence of hyperactive MAPK signaling. Similarly, we are establishing the subset of miRNAs whose activity is altered upon siRNA knockdown of Ago2, indicating that their activities are mediated specifically through Ago2, and then correlating these miRNAs with those predicted to target the MAPK gene signature. Conclusions: Hyperactive MAPK signaling is directly involved in the generation and maintenance of the ERα negative phenotype. We have shown that specific miRNAs play an important role in this biological process. Ultimately, we will determine if Ago2 plays an important role as a direct mediator of signal transduction events and global gene expression through its integral role in the biological activity of these specific miRNAs. 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 4597.