Claudin-low Breast Tumors Research Articles

Epithelial and Mesenchymal Subpopulations Within Normal Basal Breast Cell Lines Exhibit Distinct Stem Cell/Progenitor Properties

It has been proposed that epithelial-mesenchymal transition (EMT) in mammary epithelial cells and breast cancer cells generates stem cell features, and that the presence of EMT characteristics in claudin-low breast tumors reveals their origin in basal stem cells. It remains to be determined, however, whether EMT is an inherent property of normal basal stem cells, and if the presence of a mesenchymal-like phenotype is required for the maintenance of all their stem cell properties. We used nontumorigenic basal cell lines as models of normal stem cells/progenitors and demonstrate that these cell lines contain an epithelial subpopulation ("EpCAM+," epithelial cell adhesion molecule positive [EpCAM(pos)]/CD49f(high)) that spontaneously generates mesenchymal-like cells ("Fibros," EpCAM(neg)/CD49f(med/low)) through EMT. Importantly, stem cell/progenitor properties such as regenerative potential, high aldehyde dehydrogenase 1 activity, and formation of three-dimensional acini-like structures predominantly reside within EpCAM+ cells, while Fibros exhibit invasive behavior and mammosphere-forming ability. A gene expression profiling meta-analysis established that EpCAM+ cells show a luminal progenitor-like expression pattern, while Fibros most closely resemble stromal fibroblasts but not stem cells. Moreover, Fibros exhibit partial myoepithelial traits and strong similarities with claudin-low breast cancer cells. Finally, we demonstrate that Slug and Zeb1 EMT-inducers control the progenitor and mesenchymal-like phenotype in EpCAM+ cells and Fibros, respectively, by inhibiting luminal differentiation. In conclusion, nontumorigenic basal cell lines have intrinsic capacity for EMT, but a mesenchymal-like phenotype does not correlate with the acquisition of global stem cell/progenitor features. Based on our findings, we propose that EMT in normal basal cells and claudin-low breast cancers reflects aberrant/incomplete myoepithelial differentiation.

S6-5: Obesity Drives Epithelial-to-Mesenchymal Transition and Tumor Progression in a Novel Claudin-Low Mammary Cancer Model.

Abstract Background: Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative tumor initiating cell (TIC) population. However, the exact mechanism of obesity-driven tumor progression remains unknown. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. Materials, Methods, and Results: To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice, designated M-Wnt and E-Wnt. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative TIC population (62% +/−7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p<0.0001), and increased ALDH activity (7% are ALDH+, p=0.0004). M-Wnt cells have increased migration (scratch assay) and invasion in vitro (226-fold higher after 30h, p<0.0001). As few as 50 unsorted M-Wnt cells, injected into C57BL/6 mice were capable of forming a tumor. Microarray analysis revealed that M-Wnt cells display gene expression profiles virtually identical to human Claudin-low breast tumors, while E-Wnt cells clustered with basal-like tumors. EMT and stem cell gene expression patterns of M-Wnt cells were maintained in vivo, including decreased E-cadherin and increased N-cadherin, fibronectin, vimentin, SNAIL, TWIST, SLUG, FOXC2, and TGF-β (p<0.05 for all). Using these cell lines in vivo, we tested the hypothesis that energy balance modulation, through diet-induced obesity (DIO) and calorie restriction (CR), regulates the TIC population. We found that M-Wnt tumors, transplanted into ovariecomized syngeneic female C57BL/6 mice, grew at significantly different growth rates depending on the diet treatment (DIO > Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key TIC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). Discussion: In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, which demonstrates that clinical subtypes of breast cancer are differentially regulated by energy balance modulation. Additionally, our data demonstrates, for the first time, that energy balance modulation (ie, CR and obesity) directly regulates EMT, in response to local upregulation of TGF-β signaling. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S6-5.

Transgenic IGF-IR overexpression induces mammary tumors with basal-like characteristics, whereas IGF-IR-independent mammary tumors express a claudin-low gene signature

Molecular profiling has allowed a more precise classification of human cancers. With respect to breast cancer, this approach has been used to identify five subtypes; luminal A, luminal B, HER2-enriched, basal-like and claudin-low. In addition, this approach can be used to determine the type of tumor represented by particular cell lines or transgenic animal models. Therefore, this approach was utilized to classify the mammary tumors that develop in MTB-IGFIR transgenic mice. It was determined that the primary mammary tumors, which develop due to elevated expression of the type I insulin-like growth factor receptor (IGF-IR) in mammary epithelial cells, most closely resemble murine tumors with basal-like or mixed gene expression profiles and with human basal-like breast cancers. Downregulation of IGF-IR transgene in MTB-IGFIR tumor-bearing mice leads to the regression of most of the tumors, followed by tumor reappearance in some of the mice. These tumors that reappear following IGF-IR transgene downregulation do not express the IGF-IR transgene and cluster with murine mammary tumors that express a mesenchymal gene expression profile and with human claudin-low breast cancers. Therefore, IGF-IR overexpression in murine mammary epithelial cells induces mammary tumors with primarily basal-like characteristics, whereas tumors that develop following IGF-IR downregulation express a gene signature that most closely resembles human claudin-low breast tumors.

Murine mammary tumor cells with a claudin-low genotype

BackgroundMolecular classification of human breast cancers has identified at least 5 distinct tumor subtypes; luminal A, luminal B, Her2-enriched, basal-like and claudin-low. The claudin-low subtype was identified in 2007 and is characterized by low expression of luminal differentiation markers and claudins 3, 4 and 7 and high levels of mesenchymal markers. Claudin-low tumors have a reported prevalence of 7-14% and these tumors have a poor prognosis.ResultsIn this study we report the characterization of several cell lines established from mammary tumors that develop in MTB-IGFIR transgenic mice. Two lines, RM11A and RJ348 present with histological features and gene expression patterns that resemble claudin-low breast tumors. Specifically, RM11A and RJ348 cells express high levels of the mesenchymal genes Zeb1, Zeb2, Twist1 and Twist2 and very low levels of E-cadherin and claudins 3, 4 and 7. The RM11A and RJ348 cells are also highly tumorigenic when re-introduced into the mammary fat pad of mice.ConclusionsMammary tumor cells established from MTB-IGFIR transgenic mice can be used as in vitro and in vivo model systems to further our understanding of the poorly characterized, claudin-low, breast cancer subtype.

Abstract 3332: Obesity drives epithelial/mesenchymal transition and tumor progression in a novel Wnt-1 mammary cancer model

Abstract Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative breast cancer stem cell (BCSC) population. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative cancer stem cell population (62% +7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p<0.0001), and increased ALDH activity (7% are ALDH+, p=0.0004). M-Wnt cells have increased migration (scratch assay) and invasion in vitro (226-fold higher after 30h, p<0.0001). As few as 50 unsorted M-Wnt cells, injected into C57BL/6 mice were capable of forming a tumor. Microarray analysis revealed that M-Wnt cells display gene expression profiles virtually identical to human Claudin-low breast tumors, while E-Wnt cells clustered with basal-like tumors. EMT and stem cell gene expression patterns of M-Wnt cells were maintained in vivo, including decreased E-cadherin and increased N-cadherin, fibronectin, vimentin, SNAIL, TWIST, SLUG, FOXC2, and TGF-β (p<0.05 for all). Using these cell lines in vivo, we tested the hypothesis that energy balance modulation, through diet-induced obesity (DIO) and calorie restriction (CR), regulates the BCSC population. We found that M-Wnt tumors, transplanted into ovariecomized syngeneic female C57BL/6 mice, grew at significantly different growth rates depending on the diet treatment (DIO > Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key BCSC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, suggesting that energy balance modulation (ie, CR and obesity) regulates tumor growth through direct regulation of a BCSC population, possibly in response to alterations in TGF-β signaling. 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 3332. doi:10.1158/1538-7445.AM2011-3332