Plasticity Of Breast Cancer Cells Research Articles

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells

BackgroundMetastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment.MethodsHere we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages.ResultsWe found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells.ConclusionOur findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due to malignant metastases in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2411-1) contains supplementary material, which is available to authorized users.

Tumor suppressor SET9 guides the epigenetic plasticity of breast cancer cells and serves as an early-stage biomarker for predicting metastasis.

During the course of cancer progression, neoplastic cells undergo dynamic and reversible transitions between multiple phenotypic states, and this plasticity is enabled by underlying shifts in epigenetic regulation. Our results identified a negative feedback loop in which SET9 controls DNA methyltransferase-1 protein stability, which represses the transcriptional activity of the SET9 promoter in coordination with Snail. The modulation of SET9 expression in breast cancer cells revealed a connection with E2F1 and the silencing of SET9 was sufficient to complete an epigenetic program that favored epithelial-mesenchymal transition and the generation of cancer stem cells, indicating that SET9 plays a role in modulating breast cancer metastasis. SET9 expression levels were significantly higher in samples from patients with pathological complete remission than in samples from patients with disease recurrence, which indicates that SET9 acts as a tumor suppressor in breast cancer and that its expression may serve as a prognostic marker for malignancy.

Abstract 3345: Inhibition of STAT3 reduces radiation induced changes in cellular plasticity and sensitizes breast cancer cells to radiation

Abstract Triple negative breast cancer (TNBC) is categorized as a lack of expression of the hormone receptors, estrogen and progesterone, and HER2, and is a highly aggressive form of breast cancer. Due to the receptor status, TNBC is unresponsive to conventional targeted treatment resulting in a poor prognosis and increased risk of recurrence and metastasis. We have observed that TNBC patients have higher amounts of infiltrating leukocytes compared to estrogen receptor positive breast cancer patients. Therefore, inflammation may be a contributing factor in TNBC and inhibition of key inflammatory pathways may aid in sensitizing TNBC cells to treatment. Growing evidence suggests that radioresistant breast cancer stem cells are responsible for the tumor growth, recurrence, and metastasis following treatment. Previous studies have suggested that radiation alters non-stem cell populations to become more stem-like, indicating that radiation may induce changes in cellular plasticity. Cytokine activation of the signal transducer and activator of transcription 3 (STAT3) has been shown to mediate tumor-promoting inflammation and expansion of stem cell populations. Thus, we wanted to determine how inhibition of the STAT3 pathway alters the response of TNBC to radiation. To determine the effect of STAT3 inhibition on breast cancer stem populations, TNBC cells were treated with two different small molecule inhibitors to STAT3, C188-9 or Stattic, to block STAT3 signaling and the enzymatic activity of the cancer stem cell marker, aldehyde dehydrogenase (ALDH), was measured by Aldefluor assay. Treatment with 10 μM C188-9 decreased the ALDH positive (ALDH+) population from 18.3% to 5.2% and treatment with 1 μM Stattic decreased the ALDH+ from 12.2% to 4.9% indicating that inhibition of STAT3 decreases the stem cell population of TNBC. In order to determine the effect of STAT3 inhibition on radiation induced plasticity of breast cancer cells, TNBC cells were isolated into ALDH+ and ALDH negative (ALDH-) populations using flow cytometry and irradiated with 8 Gy of radiation. Following five days of treatment, both populations exhibited an increase in the percentage of ALDH+ cells indicating an increase in stem-like cells following radiation. Treatment with either 10 μM C188-9 or 1 μM Stattic significantly blocked the radiation induced increase in breast cancer stem cells. The combined effect of radiation and STAT3 inhibition on colony formation was also assessed. Exposure to 8 Gy of radiation decreased colony formation; however, inhibition of STAT3 prior to radiation exposure significantly reduced colony formation and thus sensitized the breast cancer cells to radiation treatment. These data suggest that targeting STAT3 could potentially be used to prevent alterations in cellular plasticity induced by radiation, reduce populations of resistant stem-like cells, and improve treatment outcomes. Citation Format: Kimberly M. Arnold, Lynn M. Opdenaker, Daniel Flynn, Jennifer Sims-Mourtada. Inhibition of STAT3 reduces radiation induced changes in cellular plasticity and sensitizes breast cancer cells to radiation. [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 3345. doi:10.1158/1538-7445.AM2015-3345

Somatic Cell Fusions Reveal Extensive Heterogeneity in Basal-like Breast Cancer

Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

Targeting metabolic plasticity in breast cancer cells via mitochondrial complex I modulation.

Heterogeneity commonly observed in clinical tumors stems both from the genetic diversity as well as from the differential metabolic adaptation of multiple cancer types during their struggle to maintain uncontrolled proliferation and invasion in vivo. This study aims to identify a potential metabolic window of such adaptation in aggressive human breast cancer cell lines. With a multidisciplinary approach using high-resolution imaging, cell metabolism assays, proteomic profiling and animal models of human tumor xenografts and via clinically-relevant pharmacological approach for modulating mitochondrial complex I function in human breast cancer cell lines, we report a novel route to target metabolic plasticity in human breast cancer cells. By a systematic modulation of mitochondrial function and by mitigating metabolic switch phenotype in aggressive human breast cancer cells, we demonstrate that the resulting metabolic adaptation signatures can predictably decrease tumorigenic potential in vivo. Proteomic profiling of the metabolic adaptation in these cells further revealed novel protein-pathway interactograms highlighting the importance of antioxidant machinery in the observed metabolic adaptation. Improved metabolic adaptation potential in aggressive human breast cancer cells contribute to improving mitochondrial function and reducing metabolic switch phenotype-which may be vital for targeting primary tumor growth in vivo.

242: Characterisation of retinoic acid effect on breast cancer cell plasticity

242: Characterisation of retinoic acid effect on breast cancer cell plasticity

Mitochondrial complex I modulation targets metabolic plasticity in breast cancer cells

Background Cancer cell transformation entails significant alterations in intracellular metabolic pathways one of which is the aerobic glycolysis phenotype [1]. The original formalism of this phenotype was hypothesized by Otto Warburg in 1950s and main tenet of his hypothesis was that mitochondrial dysfunction in cancer cells leads to aerobic glycolysis phenotype [2]. It is not clear in the field if mitochondrial dysfunction is a necessary condition for observing the aerobic glycolysis phenotype and further it is also not known if this “metabolic switch” phenotype is reversible. We recently showed that mitochondrial dysfunction (generated by gene silencing of catalytic subunit of mitochondrial complex I in transformed cells indeed can induce metabolic switch phenotype [3]. We further demonstrated that this can be reversed for moderate mitochondrial dysfunction models. In the present study, we ask if we can achieve tumor control in preclinical animal models by systematic modulation of mitochondrial complex I function via metabolic adaption to clinically relevant pharmacological modulators of mitochondria in breast cancer cells.

Epithelial-mesenchymal transition transcription factors and miRNAs: "Plastic surgeons" of breast cancer.

Growing evidence suggests that breast cancer cell plasticity arises due to a partial reactivation of epithelial-mesenchymal transition (EMT) programs in order to give cells pluripotency, leading to a stemness-like phenotype. A complete EMT would be a dead end program that would render cells unable to fully metastasize to distant organs. Evoking the EMT-mesenchymal-to-epithelial transition (MET) cascade promotes successful colonization of distal target tissues. It is unlikely that direct reprogramming or trans-differentiation without passing through a pluripotent stage would be the preferred mechanism during tumor progression. This review focuses on key EMT transcriptional regulators, EMT-transcription factors involved in EMT (TFs) and the miRNA pathway, which are deregulated in breast cancer, and discusses their implications in cancer cell plasticity. Cross-regulation between EMT-TFs and miRNAs, where miRNAs act as co-repressors or co-activators, appears to be a pivotal mechanism for breast cancer cells to acquire a stem cell-like state, which is implicated both in breast metastases and tumor recurrence. As a master regulator of miRNA biogenesis, the ribonuclease type III endonuclease Dicer plays a central role in EMT-TFs/miRNAs regulating networks. All these EMT-MET key regulators represent valuable new prognostic and predictive markers for breast cancer as well as promising new targets for drug-resistant breast cancers.

Dorsomorphin reverses the mesenchymal phenotype of breast cancer initiating cells by inhibition of bone morphogenetic protein signaling

Increasing evidence supports the theory that tumor growth, homeostasis, and recurrence are dependent on a small subset of cells with stem cell properties, redefined cancer initiating cells (CICs) or cancer stem cells. Bone morphogenetic proteins (BMPs) are involved in cell-fate specification during embryogenesis, in the maintenance of developmental potency in adult stem cells and may contribute to sustain CIC populations in breast carcinoma. Using the mouse A17 cell model previously related to mesenchymal cancer stem cells and displaying properties of CICs, we investigated the role of BMPs in the control of breast cancer cell plasticity. We showed that an autocrine activation of BMP signaling is crucial for the maintenance of mesenchymal stem cell phenotype and tumorigenic potential of A17 cells. Pharmacological inhibition of BMP signaling cascade by Dorsomorphin resulted in the acquisition of epithelial-like traits by A17 cells, including expression of Citokeratin-18 and E-cadherin, through downregulation of Snail and Slug transcriptional factors and Cyclooxygenase-2 (COX2) expression, and in the loss of their stem-features and self-renewal ability. This phenotypic switch compromised A17 cell motility, invasiveness and in vitro tumor growth. These results reveal that BMPs are key molecules at the crossroad between stemness and cancer.

Poised Chromatin at the ZEB1 Promoter Enables Breast Cancer Cell Plasticity and Enhances Tumorigenicity

The recent discovery that normal and neoplastic epithelial cells re-enter the stem cell state raised the intriguing possibility that the aggressiveness of carcinomas derives not from their existing content of cancer stem cells (CSCs) but from their proclivity to generate new CSCs from non-CSC populations. Here, we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find that plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration, enabling them to respond readily to microenvironmental signals, such as TGFβ. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases, and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model in which interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential.

P130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells

IntroductionIntrinsic plasticity of breast carcinoma cells allows them to undergo a transient and reversible conversion into mesenchymal cells to disseminate into distant organs, where they can re-differentiate to an epithelial-like status to form a cohesive secondary mass. The p130Cas scaffold protein is overexpressed in human ER+ and HER2+ breast cancer where it contributes to cancer progression, invasion and resistance to therapy. However, its role in regulating mesenchymal aggressive breast cancer cells remains to be determined. The aim of this study was to investigate the molecular and functional involvement of this adaptor protein in breast cancer cell plasticity.MethodsWe used silencing strategies and rescue experiments to evaluate phenotypic and biochemical changes from mesenchymal to epithelial traits in breast tumor cell lines. In the mouse A17 cell model previously related to mesenchymal cancer stem cells and basal-like breast cancer, we biochemically dissected the signaling pathways involved and performed functional in vivo tumor growth ability assays. The significance of the signaling platform was assessed in a human setting through the use of specific inhibitors in aggressive MDA-MB-231 subpopulation LM2-4175 cells. To evaluate the clinical relevance of the results, we analyzed publicly available microarray data from the Netherlands Cancer Institute and from the Koo Foundation Sun Yat-Sen Cancer Center.ResultsWe show that p130Cas silencing induces loss of mesenchymal features, by downregulating Vimentin, Snail, Slug and Twist transcriptional factors, resulting in the acquirement of epithelial-like traits. Mechanistically, p130Cas controls Cyclooxygenase-2 transcriptional expression, which in turn contributes to p130Cas-dependent maintenance of mesenchymal phenotype. This cascade of events also compromises in vivo tumor growth through inhibition of cell signaling controlling cell cycle progression. c-Src and JNK kinases are sequential players in p130Cas/ Cyclooxygenase-2 axis and their pharmacological inhibition is sufficient to downregulate Cyclooxygenase-2 leading to an epithelial phenotype. Finally, in silico microarray data analysis indicates that p130Cas and Cyclooxygenase-2 concomitant overexpression predicts poor survival and high probability of breast tumor recurrence.ConclusionsOverall, these data identify a new p130Cas/Cyclooxygenase-2 axis as a crucial element in the control of breast tumor plasticity, opening new therapeutic strategies leading to inhibition of these pathways in aggressive breast carcinoma.

Abstract 1514: The Warburg effect in stromal cells drives breast cancer cell plasticity via interleukin 6

Abstract Background: Intra-tumoral heterogeneity is an emerging concept in breast cancer biology. This concept entails the notion that sub-populations within the tumor possess different biologic features and hence vary in sensitivity to therapies. The inhibition of estrogen receptor alpha (ER), a hormone based therapy (HT), is a successful therapy for luminal A breast cancer patients. However, HT resistance, due to a variety of factors such as the tumor microenvironment, plays a crucial role in breast cancer progression. Genetic alterations in the stromal fibroblasts, such as deficiency for the tumor-suppressors (TP53 and pRB) within the tumor microenvironment, are associated with poor prognosis in breast cancer. Hypothesis: We propose that stromal cells promote the intra-tumoral heterogeneity in luminal A breast cancer. We developed an in vivo model to study the crosstalk between bone marrow stromal cells (HS27) immortalized with human papilloma virus (HPV) E6 onco-protein and a luminal A breast cancer cell line, MCF-7. Results: Here we report that HS27 cells secrete high amounts of IL-6, which operates in an autocrine and paracrine fashion. Paracrine IL-6 from HS27 cells contributes to MCF-7 cancer cell dissemination and metastasis. Autocrine IL-6 drives anaerobic glycolysis (Warburg effect) and produces high levels of lactate and H+ from HS27 cells. High levels of IL-6 and lactate from the conditioned media of HS27 cells promote the expansion of a MCF-7 cells sub-population expressing high levels of lactate shuttle MCT-1 and low levels of ER. This cancer cell population exhibits invasive and metastatic potential. Finally, we show that HPV DNA positive breast cancer lesions are more biologically aggressive, and express higher levels of IL-6, than negative ones. Conclusion: This study highlights that tumor microenvironment stressors (viral infections and oncogenes) promote a complex metabolic crosstalk (anaerobic glycolysis and oxidative phosphorylation) between stroma and cancer cells, ultimately sustaining cancer progression. Grant support: DOD W81XWH-10-1-1033 to PS, NIH R01 CA87637 to JB, PRIN KTRN38 2008 to MB Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1514. doi:1538-7445.AM2012-1514

The more things change ... the more things change: developmental plasticity of tumor-initiating mammary epithelial cells

In our haste to find and eliminate breast cancer stem cells, it appears as though we may have missed something. Contrary to current thought, a recent paper by Meyer and colleagues demonstrates developmental plasticity of breast cancer cells with respect to the CD24 cell surface marker, such that CD44pos; CD24pos and CD44pos; CD24low/- cells are able to give rise to one another in an activin/nodal-dependent manner, and that cells derived from single cells of either phenotype are capable of forming tumors as xenografts. If confirmed clinically, these data imply that simply targeting the CD44pos; CD24low/- breast cancer stem cell for breast cancer treatment may be destined to fail unless this plasticity is taken into account and prevented.

DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells

Downregulation of E-cadherin is a crucial event for epithelial to mesenchymal transition (EMT) in embryonic development and cancer progression. Using the EpFosER mammary tumour model we show that during EMT, upregulation of the transcriptional regulator deltaEF1 coincided with transcriptional repression of E-cadherin. Ectopic expression of deltaEF1 in epithelial cells was sufficient to downregulate E-cadherin and to induce EMT. Analysis of E-cadherin promoter activity and chromatin immunoprecipitation identified deltaEF1 as direct transcriptional repressor of E-cadherin. In human cancer cells, transcript levels of deltaEF1 correlated directly with the extent of E-cadherin repression and loss of the epithelial phenotype. The protein was enriched in nuclei of human cancer cells and physically associated with the E-cadherin promoter. RNA interference-mediated downregulation of deltaEF1 in cancer cells was sufficient to derepress E-cadherin expression and restore cell to cell adhesion, suggesting that deltaEF1 is a key player in late stage carcinogenesis.