Heterotypic clustering of circulating tumor cells and circulating cancer-associated fibroblasts facilitates breast cancer metastasis.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF-cCAF homotypic clusters, and in heterotypic clusters with CTCs. METHODS: In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I-IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells. RESULTS: We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is  involved in heterotypic clustering of these cells. CONCLUSION: We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.

Paget's disease of the nipple in a Her2-positive breast cancer xenograft model.

PURPOSE: Paget's disease (PD) of the breast is an uncommon disease of the nipple usually accompanied by an underlying carcinoma, often HER2 + , and accounting for 0.5-5% of all breast cancer. To date, histogenesis of PD of the breast remains controversial, as two theories-transformation and epidermotropic-have been proposed to explain this disease. Currently, animal models recapitulating PD of the nipple have not been described. METHODS: HER2-enriched DT13 breast cancer cells were injected into the mammary fat pad of NOD scid gamma null (NSG) female mice. Immunohistochemical staining and pathological studies were performed on tumor samples, and diagnosis of PD of the nipple was confirmed by expression of proteins characteristic of Paget cells (epidermal growth factor 2 (HER2), androgen receptor (AR), cytokeratin 7 (CK7), cytokeratin 8/18 (CK8/18), and mucin 1 (MUC1)). In addition, DT13 cells grown in 2D culture and in soft agar assays were sensitive to in vitro treatment with pharmacological inhibitors targeting Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. RESULTS: Mice developed tumors and nipple lesions that were detected exclusively on the tumor-bearing mammary fat pad. Tumor cells were positive for proteins characteristic of Paget cells. In vitro, DT13 cells were sensitive to inhibition of Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. CONCLUSIONS: Our results suggest that injection of HER2 + DT13 cells into the mammary fat pad of NSG mice recapitulates critical aspects of the pathophysiology of PD of the nipple, supporting the epidermotropic theory as the more likely to explain the histogenesis of this disease.

Transcriptional repression of ER through hMAPK dependent histone deacetylation by class I HDACs.

Anti-estrogen therapies are not effective in ER- breast cancers, thus identifying mechanisms underlying lack of ER expression in ER- breast cancers is imperative. We have previously demonstrated that hyperactivation of MAPK (hMAPK) downstream of overexpressed EGFR or overexpression/amplification of Her2 represses ER protein and mRNA expression. Abrogation of hMAPK in ER- breast cancer cell lines and primary cultures causes re-expression of ER and restoration of anti-estrogen responses. This study was performed to identify mechanisms of hMAPK-induced transcriptional repression of ER. We found that ER promoter activity is significantly reduced in the presence of hMAPK signaling, yet did not identify specific promoter sequences responsible for this repression. We performed an epigenetic compound screen in an ER- breast cancer cell line that expresses hMAPK yet does not exhibit ER promoter hypermethylation. A number of HDAC inhibitors were identified and confirmed to modulate ER expression and estrogen signaling in multiple ER- cell lines and tumor samples lacking ER promoter methylation. siRNA-mediated knockdown of HDACs 1, 2, and 3 reversed the mRNA repression in multiple breast cancer cell lines and primary cultures and ER promoter-associated histone acetylation increased following MAPK inhibition. These data implicate histone deacetylation downstream of hMAPK in the observed ER mRNA repression associated with hMAPK. Importantly, histone deacetylation appears to be a common mechanism in the transcriptional repression of ER between ER- breast cancers with or without ER promoter hypermethylation.

Infiltrating S100A8+ myeloid cells promote metastatic spread of human breast cancer and predict poor clinical outcome.

The mechanisms by which breast cancer (BrC) can successfully metastasize are complex and not yet fully understood. Our goal was to identify tumor-induced stromal changes that influence metastatic cell behavior, and may serve as better targets for therapy. To identify stromal changes in cancer-bearing tissue, dual-species gene expression analysis was performed for three different metastatic BrC xenograft models. Results were confirmed by immunohistochemistry, flow cytometry, and protein knockdown. These results were validated in human clinical samples at the mRNA and protein level by retrospective analysis of cohorts of human BrC specimens. In pre-clinical models of BrC, systemic recruitment of S100A8+ myeloid cells-including myeloid-derived suppressor cells (MDSCs)-was promoted by tumor-derived factors. Recruitment of S100A8+ myeloid cells was diminished by inhibition of tumor-derived factors or depletion of MDSCs, resulting in fewer metastases and smaller primary tumors. Importantly, these MDSCs retain their ability to suppress T cell proliferation upon co-culture. Secretion of macrophage inhibitory factor (MIF) activated the recruitment of S100A8+ myeloid cells systemically. Inhibition of MIF, or depletion of MDSCs resulted in delayed tumor growth and lower metastatic burden. In human BrC specimens, increased mRNA and protein levels of S100A8+ infiltrating cells are highly associated with poor overall survival and shorter metastasis free survival of BrC patients, respectively. Furthermore, analysis of nine different human gene expression datasets confirms the association of increased levels of S100A8 transcripts with an increased risk of death. Recruitment of S100A8+ myeloid cells to primary tumors and secondary sites in xenograft models of BrC enhances cancer progression independent of their suppressive activity on T cells. In clinical samples, infiltrating S100A8+ cells are associated with poor overall survival. Targeting these molecules or associated pathways in cells of the tumor microenvironment may translate into novel therapeutic interventions and benefit patient outcome.

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.

Hotspot ESR1 Mutations Are Multimodal and Contextual Modulators of Breast Cancer Metastasis.

Constitutively active estrogen receptor α (ER/ESR1) mutations have been identified in approximately one-third of ER+ metastatic breast cancers. Although these mutations are known as mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant but not local recurrences in five independent breast cancer cohorts. In concordance with transcriptomic profiling of ESR1-mutant tumors, genome-edited ESR1 Y537S and D538G-mutant cell models exhibited a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally conferred enhanced cell-cell contacts while decreasing cell-extracellular matrix adhesion. In vivo studies showed ESR1-mutant cells were associated with larger multicellular circulating tumor cell (CTC) clusters with increased compactness compared with ESR1 wild-type CTCs. These preclinical findings translated to clinical observations, where CTC clusters were enriched in patients with ESR1-mutated metastatic breast cancer. Conversely, context-dependent migratory phenotypes revealed cotargeting of Wnt and ER as a vulnerability in a D538G cell model. Mechanistically, mutant ESR1 exhibited noncanonical regulation of several metastatic pathways, including secondary transcriptional regulation and de novo FOXA1-driven chromatin remodeling. Collectively, these data provide evidence for ESR1 mutation-modulated metastasis and suggest future therapeutic strategies for targeting ESR1-mutant breast cancer. SIGNIFICANCE: Context- and allele-dependent transcriptome and cistrome reprogramming in mutant ESR1 cell models elicit diverse metastatic phenotypes related to cell adhesion and migration, which can be pharmacologically targeted in metastatic breast cancer.

Simultaneous measurement of ERα, HER2, and phosphoERK1/2 in breast cancer cell lines by flow cytometry.

The activation of human epidermal growth factor receptor-2 (HER2) results in the activation of the mitogen-activated protein kinase (MAPK) cascade that may lead to the resistance to anti-estrogen therapy in estrogen receptor (ERα) expressing breast cancer by means of phosphorylation of ERα in the N-terminal region by phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and by means of decreasing ERα expression. Immunohistochemistry is the most widely used technique for the detection of ERα and HER2 in breast cancer specimens, however, is inadequate in its ability to assess the relationship between ERα, HER2, and MAPK cascade at the single cell level. To clear this major hurdle, we devised a novel flow cytometric method to quantify the expression of ERα, HER2, and the activation of MAPK cascade simultaneously in single cells. The method was validated by concurrent Western blotting in established cell lines: MDA-231 (ERα and HER2-negative), MCF-7 (ERα-positive, HER2-negative), MCF-7 cells overexpressing ERα after long-term incubation in estrogen-free medium, and HER2 transfected MCF7 cells. Using the flow cytometry method, we confirmed the previous finding that ERα expression is down-regulated upon epidermal growth factor mediated ERK1/2 phosphorylation in EGFR/MCF-7 cells. To our knowledge, this is the first such assay to incorporate simultaneous single cell measurement for all of these pathways, which may prove useful to determine the intratumoral heterogeneity in breast tumors or the receptor status in circulating tumor cells.

Improved prognostic classification of breast cancer defined by antagonistic activation patterns of immune response pathway modules.

BACKGROUND: Elucidating the activation pattern of molecular pathways across a given tumour type is a key challenge necessary for understanding the heterogeneity in clinical response and for developing novel more effective therapies. Gene expression signatures of molecular pathway activation derived from perturbation experiments in model systems as well as structural models of molecular interactions ("model signatures") constitute an important resource for estimating corresponding activation levels in tumours. However, relatively few strategies for estimating pathway activity from such model signatures exist and only few studies have used activation patterns of pathways to refine molecular classifications of cancer. METHODS: Here we propose a novel network-based method for estimating pathway activation in tumours from model signatures. We find that although the pathway networks inferred from cancer expression data are highly consistent with the prior information contained in the model signatures, that they also exhibit a highly modular structure and that estimation of pathway activity is dependent on this modular structure. We apply our methodology to a panel of 438 estrogen receptor negative (ER-) and 785 estrogen receptor positive (ER+) breast cancers to infer activation patterns of important cancer related molecular pathways. RESULTS: We show that in ER negative basal and HER2+ breast cancer, gene expression modules reflecting T-cell helper-1 (Th1) and T-cell helper-2 (Th2) mediated immune responses play antagonistic roles as major risk factors for distant metastasis. Using Boolean interaction Cox-regression models to identify non-linear pathway combinations associated with clinical outcome, we show that simultaneous high activation of Th1 and low activation of a TGF-beta pathway module defines a subtype of particularly good prognosis and that this classification provides a better prognostic model than those based on the individual pathways. In ER+ breast cancer, we find that simultaneous high MYC and RAS activity confers significantly worse prognosis than either high MYC or high RAS activity alone. We further validate these novel prognostic classifications in independent sets of 173 ER- and 567 ER+ breast cancers. CONCLUSION: We have proposed a novel method for pathway activity estimation in tumours and have shown that pathway modules antagonize or synergize to delineate novel prognostic subtypes. Specifically, our results suggest that simultaneous modulation of T-helper differentiation and TGF-beta pathways may improve clinical outcome of hormone insensitive breast cancers over treatments that target only one of these pathways.

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

Suppression of Tumor Growth, Metastasis, and Signaling Pathways by Reducing FOXM1 Activity in Triple Negative Breast Cancer.

Metastasis-related complications account for the overwhelming majority of breast cancer mortalities. Triple negative breast cancer (TNBC), the most aggressive breast cancer subtype, has a high propensity to metastasize to distant organs, leading to poor patient survival. The forkhead transcription factor, FOXM1, is especially upregulated and overexpressed in TNBC and is known to regulate multiple signaling pathways that control many key cancer properties, including proliferation, invasiveness, stem cell renewal, and therapy resistance, making FOXM1 a critical therapeutic target for TNBC. In this study, we test the effectiveness of a novel class of 1,1-diarylethylene FOXM1 inhibitory compounds in suppressing TNBC cell migration, invasion, and metastasis using in vitro cell culture and in vivo tumor models. We show that these compounds inhibit the motility and invasiveness of TNBC MDA-MB-231 and DT28 cells, along with reducing the expression of important epithelial to mesenchymal transition (EMT) associated genes. Further, orthotopic tumor studies in NOD-SCID-gamma (NSG) mice demonstrate that these compounds reduce FOXM1 expression and suppress TNBC tumor growth as well as distant metastasis. Gene expression and protein analyses confirm the decreased levels of EMT factors and FOXM1-regulated target genes in tumors and metastatic lesions in the inhibitor-treated animals. The findings suggest that these FOXM1 suppressive compounds may have therapeutic potential in treating triple negative breast cancer, with the aim of reducing tumor progression and metastatic outgrowth.

Liquid biopsy: expanding the frontier of circulating biomarker discovery and validation in breast cancer.

Liquid biopsies represent an attractive, minimally-invasive alternative to surgical sampling or complex imaging of breast cancer and breast cancer metastasis. Here we present a summary of the major biomarker components often evaluated in liquid biopsy samples from patients with breast cancer, including circulating tumor cells, circulating cell-free tumor DNA, and cancer-associated plasma proteins. We discuss recent advancements in methods of detection and use of these biomarkers in breast cancer. Finally, we highlight some of our own recent contributions to breast cancer liquid biopsy, including the identification and characterization of circulating Cancer Associated Fibroblasts.

Suppression of FOXM1 activities and breast cancer growth in vitro and in vivo by a new class of compounds.

The transcription factor FOXM1 is upregulated and overexpressed in aggressive, therapy-resistant forms of hormone receptor-positive and triple negative breast cancers, and is associated with less good patient survival. FOXM1 signaling is also a key driver in many other cancers. Here, we identify a new class of compounds effective in suppressing FOXM1 activity in breast cancers, and displaying good potency for antitumor efficacy. The compounds bind directly to FOXM1 and alter its proteolytic sensitivity, reduce the cellular level of FOXM1 protein by a proteasome- dependent process, and suppress breast cancer cell proliferation and cell cycle progression and increase apoptosis. RNA-seq and gene set enrichment analyses indicate that the compounds decrease expression of FOXM1-regulated genes and suppress gene ontologies under FOXM1 regulation. Several compounds have favorable pharmacokinetic properties and show good tumor suppression in preclinical breast tumor models. These compounds may be suitable for further clinical evaluation in targeting aggressive breast cancers driven by FOXM1.

Reversal of the estrogen receptor negative phenotype in breast cancer and restoration of antiestrogen response.

PURPOSE: In breast cancer, the presence of estrogen receptor alpha (ER) denotes a better prognosis and response to antiestrogen therapy. Lack of ERalpha correlates with overexpression of epidermal growth factor receptor or c-erbB-2. We have shown that hyperactivation of mitogen-activated protein kinase (MAPK) directly represses ERalpha expression in a reversible manner. In this study, we determine if inhibition of MAPK in established ERalpha(-) breast cancer cell lines and tumors results in reexpression of ERalpha, and further, if reexpression of ERalpha in these ERalpha(-) tumors and cell lines could restore antiestrogen responses. EXPERIMENTAL DESIGN: Established ERalpha(-) breast cancer cell lines, ERalpha(-) breast tumors, and tumor cell cultures obtained from ERalpha(-) tumors were used in this study. Inhibition of hyperactive MAPK was accomplished via the MAPK/ERK kinase 1/2 inhibitor U0126 or via upstream inhibition with Iressa or Herceptin. Western blotting or reverse transcription-PCR for ERalpha was used to assess the reexpression of ERalpha in cells treated with U0126. Growth assays with WST-1 were done to assess restoration of antiestrogen sensitivity in these cells. RESULTS: Inhibition of MAPK activity in ERalpha(-) breast cancer cell lines results in reexpression of ERalpha; upstream inhibition via targeting epidermal growth factor receptor or c-erbB-2 is equally effective. Importantly, this reexpressed ERalpha can now mediate an antiestrogen response in a subset of these ERalpha(-) breast cancer cell lines. Treatment of ERalpha(-) tumor specimens with MAPK inhibitors results in restoration of ERalpha mRNA, and similarly in epithelial cultures from ERalpha(-) tumors, MAPK inhibition restores both ERalpha protein and antiestrogen response. CONCLUSIONS: These data show both the possibility of restoring ERalpha expression and antiestrogen responses in ERalpha(-) breast cancer and suggest that there exist ERalpha(-) breast cancer patients who would benefit from a combined MAPK inhibition/hormonal therapy.

Activation of mitogen-activated protein kinase in estrogen receptor alpha-positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor alpha-negative human breast tumors.

Breast cancer presents as either estrogen receptor alpha (ERalpha) positive or negative, with ERalpha+ tumors responding to antiestrogen therapy and having a better prognosis. By themselves, mRNA expression signatures of estrogen regulation in ERalpha+ breast cancer cells do not account for the vast molecular differences observed between ERalpha+ and ERalpha- cancers. In ERalpha- tumors, overexpression of epidermal growth factor receptor (EGFR) or c-erbB-2, leading to increased growth factor signaling, is observed such that mitogen-activated protein (MAP) kinase (MAPK) is significantly hyperactivated compared with ERalpha+ breast cancer. In ERalpha+/progesterone receptor-positive, estrogen-dependent MCF-7 breast cancer cells, we stably overexpressed EGFR or constitutively active erbB-2, Raf, or MAP/extracellular signal-regulated kinase kinase, resulting in cell lines exhibiting hyperactivation of MAPK, estrogen-independent growth, and the reversible down-regulation of ERalpha expression. By global mRNA profiling, we found a "MAPK signature" of approximately 400 genes consistently up-regulated or down-regulated in each of the MAPK+ cell lines. In several independent profile data sets of human breast tumors, the in vitro MAPK signature was able to accurately distinguish ER+ from ER- tumors. In addition, our in vitro mRNA profile data revealed distinct mRNA signatures specific to either erbB-2 or EGFR activation. A subset of breast tumor profiles was found to share extensive similarities with either the erbB-2-specific or the EGFR-specific signatures. Our results confirm that increased MAPK activation causes loss of ERalpha expression and suggest that hyperactivation of MAPK plays a role in the generation of the ERalpha- phenotype in breast cancer. These MAPK+ cell lines are excellent models for investigating the underlying mechanisms behind the ERalpha- phenotype.

Corrigendum: Hyaluronan, Cancer-Associated Fibroblasts and the Tumor Microenvironment in Malignant Progression.

[This corrects the article DOI: 10.3389/fcell.2018.00048.].

Hyaluronan, Cancer-Associated Fibroblasts and the Tumor Microenvironment in Malignant Progression.

This review summarizes the roles of CAFs in forming a "cancerized" fibrotic stroma favorable to tumor initiation and dissemination, in particular highlighting the functions of the extracellular matrix component hyaluronan (HA) in these processes. The structural complexity of the tumor and its host microenvironment is now well appreciated to be an important contributing factor to malignant progression and resistance-to-therapy. There are multiple components of this complexity, which include an extensive remodeling of the extracellular matrix (ECM) and associated biomechanical changes in tumor stroma. Tumor stroma is often fibrotic and rich in fibrillar type I collagen and hyaluronan (HA). Cancer-associated fibroblasts (CAFs) are a major source of this fibrotic ECM. CAFs organize collagen fibrils and these biomechanical alterations provide highways for invading carcinoma cells either under the guidance of CAFs or following their epithelial to mesenchymal transition (EMT). The increased HA metabolism of a tumor microenvironment instructs carcinoma initiation and dissemination by performing multiple functions. The key effects of HA reviewed here are its role in activating CAFs in pre-malignant and malignant stroma, and facilitating invasion by promoting motility of both CAFs and tumor cells, thus facilitating their invasion. Circulating CAFs (cCAFs) also form heterotypic clusters with circulating tumor cells (CTC), which are considered to be pre-cursors of metastatic colonies. cCAFs are likely required for extravasation of tumors cells and to form a metastatic niche suitable for new tumor colony growth. Therapeutic interventions designed to target both HA and CAFs in order to limit tumor spread and increase response to current therapies are discussed.

Circulating Tumor Cells: Strategies for Capture, Analyses, and Propagation.

Circulating tumor cells (CTCs) play a central role in tumor dissemination and metastases, which are ultimately responsible for most cancer deaths. Technologies that allow for identification and enumeration of rare CTC from cancer patients' blood have already established CTC as an important clinical biomarker for cancer diagnosis and prognosis. Indeed, current efforts to robustly characterize CTC as well as the associated cells of the tumor microenvironment such as circulating cancer associated fibroblasts (cCAF), are poised to unmask key insights into the metastatic process. Ultimately, the clinical utility of CTC will be fully realized once CTC can be reliably cultured and proliferated as a biospecimen for precision management of cancer patients, and for discovery of novel therapeutics. In this review, we highlight the latest CTC capture and analyses technologies, and discuss in vitro strategies for culturing and propagating CTC.

MAPK Activation Predicts Poor Outcome and the MEK Inhibitor, Selumetinib, Reverses Antiestrogen Resistance in ER-Positive High-Grade Serous Ovarian Cancer.

PURPOSE: Although 67% of high-grade serous ovarian cancers (HGSOC) express the estrogen receptor (ER), most fail antiestrogen therapy. Because MAPK activation is frequent in ovarian cancer, we investigated if estrogen regulates MAPK and if MEK inhibition (MEKi) reverses antiestrogen resistance. EXPERIMENTAL DESIGN: Effects of MEKi (selumetinib), antiestrogen (fulvestrant), or both were assayed in ER-positive HGSOC in vitro and in xenografts. Response biomarkers were investigated by gene expression microarray and reverse phase protein array (RPPA). Genes differentially expressed in two independent primary HGSOC datasets with high versus low pMAPK by RPPA were used to generate a "MAPK-activated gene signature." Gene signature components that were reversed by MEKi were then identified. RESULTS: High intratumor pMAPK independently predicts decreased survival (HR, 1.7; CI > 95%,1.3-2.2; P = 0.0009) in 408 HGSOC from The Cancer Genome Atlas. A differentially expressed "MAPK-activated" gene subset was also prognostic. "MAPK-activated genes" in HGSOC differ from those in breast cancer. Combined MEK and ER blockade showed greater antitumor effects in xenografts than monotherapy. Gene set enrichment analysis and RPPA showed that dual therapy downregulated DNA replication and cell-cycle drivers, and upregulated lysosomal gene sets. Selumetinib reversed expression of a subset of "MAPK-activated genes" in vitro and/or in xenografts. Three of these genes were prognostic for poor survival (P = 0.000265) and warrant testing as a signature predictive of MEKi response. CONCLUSIONS: High pMAPK is independently prognostic and may underlie antiestrogen failure. Data support further evaluation of fulvestrant and selumetinib in ER-positive HGSOC. The MAPK-activated HGSOC signature may help identify MEK inhibitor responsive tumors.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b-Mediated Malignant Progression.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.

Breast cancer, stem/progenitor cells and the estrogen receptor.

Recently, substantial progress has been made in the identification and characterization of stem and progenitor cells in the mouse and human mammary gland. Furthermore, there is accumulating evidence that these cells might be targets for transformation during mammary carcinogenesis. On the basis of this stem cell concept, we propose a model in which the transformation of different subsets of stem and progenitor cells results in the diversity of breast cancer phenotypes, including expression of the estrogen receptor in breast cancers subtypes. This model has important implications for understanding mammary carcinogenesis. Furthermore, the concept of breast cancer as a disease of mammary stem and progenitor cells has profound implications for the development of new strategies for breast cancer prevention and therapy.