Xenograft-Derived Human Breast Cancer Organoids Can Form Chimeras with Host Mouse Mammary Epithelial Cells Which Promote Tumor Cell Proliferation

Breast cancer is the second leading cause of cancer deaths in the United States. At the present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display ‘normal’ behavior when placed into ‘normal’ ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Human metastatic (MDA-MB-231-GFP), non-metastatic (MDA-MB-468-GFP), and metastatsis-suppressed (MDA-MB-231BRMS-GFP) breast cancer cells were mixed with mouse mammary epithelial cells at ratios of 1:5 (MDA-231-GFP) or 1:50 (MDA-468-GFP, MDA-231BRMS-GFP) in phosphate-buffered saline. Ten microliters of the solution was injected into mammary fat pads cleared of host epithelium of 3-week-old female athymic nude mice. Immortalized human mammary epithelial cells (hTERT-HME1; 50K) and mouse mammary epithelial cells (50K) were independently inoculated into epithelium-free fat pads as controls. Ten to twelve weeks later, mice were euthanized and their fat pad outgrowths harvested for histological examination and immunostaining for mammary epithelial cell markers, keratin 14 and casein (human vs. mouse). Tissue fragments excised from first-generation fat pad outgrowths were implanted into the cleared mammary fat pads of a new group of 3-week-old female athymic nude mice to produce second-generation outgrowths. When MDA-MB-231-GFP metastatic, MDA-MB-231BRMS1-GFP metastasis-suppressed, and MDA-MB-468-GFP non-metastatic breast cancer cells were mixed with mouse mammary epithelial cells and inoculated into mammary fat pads of mice cleared of epithelium, no primary tumors or bone metastases were formed, and the human breast cancer cells 1) contributed to the formation of the regenerated mouse mammary gland and 2) expressed the human mammary epithelial marker keratin 14 in consecutive sections of first generation transplants. Furthermore, mouse mammary epithelial cells expressing mouse keratin 14 were found juxtaposed to human cancer cells expressing human keratin 14. Overall, these data suggest that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells can be redirected towards a non-tumorigenic/non-metastatic phenotype by signals produced by the regenerating mouse mammary gland. Supported by the National Institutes of Health, National Cancer Institute Intramural Program. 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 434. doi:10.1158/1538-7445.AM2011-434

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

In Vivo Evidence for the Role of CD44s in Promoting Breast Cancer Metastasis to the Liver

Cancer stem cells (CSCs) are small portions of cancer cells that have important self-renewal and proliferation characteristics, and therefore, they play a significant role in cancer development and metastasis. Recently, several studies have revealed CSCs of several solid tumors. The identification and targeting of CSCs are expected to advance treatment outcomes, but until now, only a few drugs that specifically target CSCs have been developed. Metformin, an antidiabetic drug used for the treatment of type 2 diabetes mellitus, suppresses gluconeogenesis in the liver and increases insulin sensitivity and glucose uptake in peripheral tissues such as muscles. Moreover, the drug has shown an anti-cancer effect by inhibiting cellular proliferation and protein synthesis in several cancer cell lines. Additionally, many clinical and epidemiological studies have suggested that metformin might be associated with a decreased risk of cancer development and an increased response to chemotherapy. In addition to its anticarcinogenic effect, studies about the anti-cancer stem cell effect of metformin have been recently reported. Several reports revealed that metformin could preferentially kill cancer stem cells, including breast cancer stem cells. However, the mechanism of anti-cancer stem cell activity is not well known. Therefore, this study was planned to confirm the cytotoxicity of metformin, especially for breast cancer stem cells as well as breast cancer cells. To this end, several human breast cancer cell lines and normal mouse mammary epithelial cell lines were used. In this study, metformin showed cell growth inhibition in human breast cancer cells and mouse mammary epithelial cells by inducing cell cycle arrest and apoptosis. Moreover, metformin specifically killed breast cancer stem cells. Metformin showed reduced mammosphere formation in all cell lines. In addition, decreasing breast cancer stem cell population by metformin treatment was identified through FACS analysis. For the mechanism of anti-cancer stem cell activity, western blot assay was performed. We identified decreased levels of cyclin D and cycline E and increased levels of Bax in secondary mammaospheres according to metformin treatment. In conclusion, this present study show that metformin kills breast cancer stem cells as well as breast cancer cells, and these phenomena occur by the induction of cell cycle arrest and apoptosis. Citation Format: Eun Joo Kang, Jae Hong Seo, Jun Suk Kim, Ji Young Kim. Metformin represses stem cell properties and induces apoptosis in human breast cancer cells and mouse mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 208. doi:10.1158/1538-7445.AM2014-208

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and β (ERβ)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERβ expression (T47D-ERβ), the effect of a varying intracellular ERα/ERβ ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ERα-expressing T47D-ERβ cells with inhibited ERβ expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ERβ breast cancer cells with low levels of ERα and no expression of ERβ. In addition, data from our study indicate that ERβ-mediated gene and protein expression counteracts ERα-mediated effects because in T47D-ERβ cells expressing ERβ and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ERβ decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ERβ cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ERα/ERβ ratio) in the cells or tissue of interest.

Selection of Brain Metastasis-Initiating Breast Cancer Cells Determined by Growth on Hard Agar

Folic acid-tripeptide-conjugated synthetic biodegradable nanoparticle-loaded with Ormeloxifene potentially inhibited breast cancer xenograft tumor

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Breast and colorectal cancers are among the most common tumor types worldwide and the occurrence of metastases is often associated with a shortened lifespan. One of the signaling pathways that has been frequently associated with metastasis in these tumor entities is the Wnt signaling pathway. Wnt signaling can be either β-catenin dependent (canonical) or βcatenin independent (non-canonical). In breast and colorectal cancer, tumor-promoting properties could be attributed to members of the non-canonical Wnt signaling pathway. Preliminary results showed that overexpression of ROR2 as a non-canonical Wnt receptor, mediated an aggressive phenotype in breast cancer cells and could significantly increase invasion. Accordingly, the first aim of this work was to investigate which ligand binds ROR2 and thus triggers the invasive behavior of MCF-7 cells. RNA-seq analysis revealed increased expression levels of the non-canonical ligand Wnt11 in ROR2 overexpressing cells. Hence, Wnt11 was further investigated as a potential ROR2-ligand. Using co-immunoprecipitation experiments, this work demonstrated the interaction of ROR2 with Wnt11 in human breast cancer cells. To determine which domain facilitates the ROR2-mediated invasion, sequential deletions of the different ROR2 domains were induced. This demonstrated that the cysteine-rich and the tyrosine kinase domain mediate this effect. The next step was to determine whether Wnt11 binds other receptors that trigger the invasive behavior of MCF-7 cells since ROR2 is not expressed in these cells. Furthermore, a cell line screening of different breast and colorectal cancer cell lines identified FZD4 and FZD6 as highly expressed non-canonical Wnt receptors. An interaction between Wnt11 and FZD6 was validated by using Co-IP, in which PTK7 appears to act as a co-receptor. To investigate functional implications of Wnt11-mediated signaling in breast cancer, MCF-7 cells were stimulated with recombinant Wnt11, which caused increased invasion and migration rates, whereas loss of FZD6 resulted in a significant decrease of the elevated invasion rates. In line with the collected data, a signature with non-canonical Wnt pathway members including FZD receptors, ROR receptors, Wnt ligands and PI3K signaling members was defined and was analyzed for its DMFS prognostic values in breast and colorectal cancer patients. Therefore, the signature was applied to gene expression data of primary breast and colorectal cancer patients. For the primary breast cancer patients, the signature clustered the data set into two patient groups, among which the group with high Wnt11 expression was associated with poor DMFS. Considering FZD4 and FZD6 individually, breast cancer patients with high FZD6 gene expression showed worse DMFS, while high FZD4 levels were associated with favorable DMFS. Interestingly, the defined signature clustered the data set of the colorectal cancer patients into four groups and the cohort with a high FZD6 expression exhibited a poor DMFS compared to the other groups. Another important aspect of tumor progression is the reprogramming of the tumor microenvironment. It has been shown that tumor-derived extracellular vesicles can influence the tumor microenvironment in different ways. Preliminary data demonstrated that Wnt proteins can be transported via extracellular vesicles to target cells and induce Wnt signaling responses there. It was shown that RORs are transported on microvesicles and exosomes. Modulation of ROR1 and ROR2 expression resulted in altered protein compositions for both vesicle populations. However, no major impact on vesicle size or concentration was evident. Further analysis addressed functional consequences of ROR1 and ROR2 expression on extracellular vesicles. Tumor-derived EVs isolated from the aggressive breast cancer cell line MDA-MB231 induced invasiveness in MCF-7 cells, which was shown to be dependent on vesicular ROR1 expression. To determine whether ROR1/2 can function as a biomarker in breast cancer patients, plasma-derived microvesicles were analyzed for their ROR1 and ROR2 expression by flow cytometry, which is currently ongoing. In conclusion, this work demonstrated the importance of non-canonical Wnt signaling in breast cancer progression. Wnt11 has been identified as a novel ligand for ROR2 and FZD6 by co-immunoprecipitation experiments, thereby mediating tumor-promoting properties in breast cancer. In particular, it has been shown that ROR proteins not only play an important role in breast cancer cells themselves, but also appear to be additionally involved in vesicle biogenesis and can furthermore be transferred to target cells. A clinical applicability of both ROR proteins regarding their usage as tumor biomarkers for breast cancer is still under investigation.

Spontaneous Formation of Tumorigenic Hybrids between Breast Cancer and Multipotent Stromal Cells Is a Source of Tumor Heterogeneity

The identification of new therapeutic targets and treatments to reduce tumor metastasis homing requires alternative interrogation approaches. The roles of the chemokine CCL5 and its receptor CCR5 in breast cancer progression are controversial. Cancer metastasis is regulated by chemokines in the microenvironment. Chemokines bind to cell surface receptors that belong to the G-protein-coupled receptor family (GPCRs), controlling diverse biological and pathological processes from immune surveillance, inflammation, and cancer. Previous studies of human breast cancer and breast cancer cell lines demonstrated that the chemokine receptors CXCR4 and CCR7 are expressed in breast cancer cells, malignant breast tumors, and metastasis. Their related ligands, CXCL12 (SDF1) and CCL21, are also expressed at the site of metastasis. Subsequent studies identified altered expression of CCL5 (RANTES) in breast cancer patients, correlating with disease progression. CCL5 can be expressed and secreted either by breast cancer cells or by non-malignant stromal cells at the primary or metastatic sites. However, the roles of CCL5 and its receptors in breast cancer are not fully understood. CCL5 facilitates disease progression by recruiting and modulating the activity of inflammatory cells, which subsequently remodel the tumor microenvironment. Accordingly, inhibition of CCR5 by a peptide antagonist reduced leukocyte infiltration and reduced tumor growth after subcutaneous injection of 410.4 mammary carcinoma cells into immunocompetent mice. Our recent microarray analysis of 2,254 human breast cancers demonstrated increased expression of CCL5 and its receptor CCR5, but not CCR3, in the basal and HER-2 genetic subtypes of breast cancer. Interrogation of pathways activated in patient normal breast vs. tumor identified up regulation of a CCR5 signaling module. At the same time, we also extended our research to prostate cancers. Using isogenic oncogene transformed breast and prostate cancer cell lines we show oncogene transformation induces CCR5 expression in breast and prostate epithelial cells. Further we show that the subpopulation of cells that express functional CCR5 display increased invasiveness. Studies in vivo demonstrated that CCR5 promoted metastasis homing. The FDA approved CCR5 antagonists Maraviroc or Vicriviroc, developed to block CCR5 HIV co-receptor function, reduced in vitro invasion of basal breast cancer and prostate cancer cell lines without affecting cell proliferation or viability. In a series of preclinical mouse models, used at equivalent doses to those used in treatment of humans for HIV, Maraviroc decreased breast pulmonary metastasis. The isogenic prostate cancer cell lines metastasized to bones in immune-competent mice representing an ideal model for testing anti-metastasis therapies. CCR5 was expressed in the metastasis in the bones. Maraviroc reduced prostate cancer metastasis to brain, bones and lungs. Our findings provide evidence for a key role of CCL5/CCR5 in the metastasis of basal breast cancer and prostate cancer cell lines and suggest that CCR5 antagonists may be used as an adjuvant therapy to reduce the risk of metastasis in patients with the basal breast cancer subtype and prostate cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-04.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

Substantial data from clinical trials and epidemiological studies show promising results for use of statins in many cancers, including mammary carcinoma. Breast tumor primarily metastasizes to bone to form osteolytic lesions, causing severe pain and pathological fracture. Here, we report that simvastatin acts as an inhibitor of osteolysis in a mouse model of breast cancer skeletal metastasis of human mammary cancer cell MDA-MB-231, which expresses the mutant p53R280K. Simvastatin and lovastatin attenuated migration and invasion of MDA-MB-231 and BT-20 breast tumor cells in culture. Acquisition of phenotype to express the cancer stem cell marker, CD44, leads to invasive potential of the tumor cells. Interestingly, statins significantly decreased the expression of CD44 protein via a transcriptional mechanism. shRNA-mediated down-regulation of CD44 markedly reduced the migration and invasion of breast cancer cells in culture. We identified that in the MDA-MB-231 cells, simvastatin elevated the levels of mutated p53R280K, which was remarkably active as a transcription factor. shRNA-derived inhibition of mutant p53R280K augmented the expression of CD44, leading to increased migration and invasion. Finally, we demonstrate an inverse correlation between expression of p53 and CD44 in the tumors of mice that received simvastatin. Our results reveal a unique function of statins, which foster enhanced expression of mutant p53R280K to prevent breast cancer cell metastasis to bone.

BackgroundThe development of multi-drug resistant (MDR) cancer is a significant challenge in the clinical treatment of recurrent disease. Hypoxia is an environmental selection pressure that contributes to the development of MDR. Many cancer cells, including MDR cells, resort to glycolysis for energy acquisition. This study aimed to explore the relationship between hypoxia, glycolysis, and MDR in a panel of human breast and ovarian cancer cells. A second aim of this study was to develop an orthotopic animal model of MDR breast cancer.MethodsNucleic and basal protein was extracted from a panel of human breast and ovarian cancer cells; MDR cells and cells pre-exposed to either normoxic or hypoxic conditions. Western blotting was used to assess the expression of MDR markers, hypoxia inducible factors, and glycolytic proteins. Tumor xenografts were established in the mammary fat pad of nu/nu mice using human breast cancer cells that were pre-exposed to either hypoxic or normoxic conditions. Immunohistochemistry was used to assess the MDR character of excised tumors.ResultsHypoxia induces MDR and glycolysis in vitro, but the cellular response is cell-line specific and duration dependent. Using hypoxic, triple-negative breast cancer cells to establish 100 mm3 tumor xenografts in nude mice is a relevant model for MDR breast cancer.ConclusionHypoxic pre-conditiong and xenografting may be used to develop a multitude of orthotopic models for MDR cancer aiding in the study and treatment of the disease.