Mouse Model Of Mammary Tumorigenesis Research Articles

Absence of integrin \u03b13\u03b21 promotes\xa0the progression of HER2-driven breast cancer in vivo

BackgroundHER2-driven breast cancer is correlated with poor prognosis, especially during its later stages. Numerous studies have shown the importance of the integrin α3β1 during the initiation and progression of breast cancer; however, its role in this disease is complex and often opposite during different stages and in different types of tumors. In this study, we aim to elucidate the role of integrin α3β1 in a genetically engineered mouse model of HER2-driven mammary tumorigenesis.MethodsTo investigate the role of α3β1 in HER2-driven tumorigenesis in vivo, we generated a HER2-driven MMTV-cNeu mouse model of mammary tumorigenesis with targeted deletion of Itga3 (Itga3 KO mice). We have further used several established triple-negative and HER2-overexpressing human mammary carcinoma cell lines and generated ITGA3-knockout cells to investigate the role of α3β1 in vitro. Invasion of cells was assessed using Matrigel- and Matrigel/collagen I-coated Transwell assays under static or interstitial fluid flow conditions. The role of α3β1 in initial adhesion to laminin and collagen was assessed using adhesion assays and immunofluorescence.ResultsTumor onset in mice was independent of the presence of α3β1. In contrast, the depletion of α3β1 reduced the survival of mice and increased tumor growth and vascularization. Furthermore, Itga3 KO mice were significantly more likely to develop lung metastases and had an increased metastatic burden compared to WT mice. In vitro, the deletion of ITGA3 caused a significant increase in the cellular invasion of HER2-overexpressing SKBR3, AU565, and BT474 cells, but not of triple-negative MDA-MB-231. This invasion suppressing function of α3β1 in HER2-driven cells depended on the composition of the extracellular matrix and the interstitial fluid flow.ConclusionDownregulation of α3β1 in a HER2-driven mouse model and in HER2-overexpressing human mammary carcinoma cells promotes progression and invasiveness of tumors. The invasion-suppressive role of α3β1 was not observed in triple-negative mammary carcinoma cells, illustrating the tumor type-specific and complex function of α3β1 in breast cancer.

HER2-Driven Breast Tumorigenesis Relies upon Interactions of the Estrogen Receptor with Coactivator MED1.

Studies of the estrogen receptor (ER) coactivator protein Mediator subunit 1 (MED1) have revealed its specific roles in pubertal mammary gland development and potential contributions to breast tumorigenesis, based on coamplification of MED1 and HER2 in certain breast cancers. In this study, we generated a mouse model of mammary tumorigenesis harboring the MMTV-HER2 oncogene and mutation of MED1 to evaluate its role in HER2-driven tumorigenesis. MED1 mutation in its ER-interacting LxxLL motifs was sufficient to delay tumor onset and to impair tumor growth, metastasis, and cancer stem-like cell formation in this model. Mechanistic investigations revealed that MED1 acted directly to regulate ER signaling through the downstream IGF1 pathway but not the AREG pathway. Our findings show that MED1 is critical for HER2-driven breast tumorigenesis, suggesting its candidacy as a disease-selective therapeutic target.Significance: These findings identify an estrogen receptor-binding protein as a critical mediator of HER2-driven breast tumorigenesis, suggesting its candidacy as a disease-selective therapeutic target. Cancer Res; 78(2); 422-35. ©2017 AACR.

Abstract A17: The CEBPD transcription factor: A signaling hub for promotion of cancer cell stemness

Abstract Our previous studies showed that the transcription factor CCAAT/enhancer binding protein delta (CEBPD) promotes tumor metastasis in a mouse model of mammary tumorigenesis (MMTV-Neu) (Balamurugan et al., EMBO J, 2010). Here we show that CEBPD serves as a signaling hub, which integrates and amplifies signaling pathways that promote stemness in breast cancer and glioblastoma as well as in embryonic carcinoma stem cells. This work identifies a mechanism by which hypoxia and inflammation promote cancer stem-like cells (CSCs), which are implicated in tumor progression, metastasis, and therapy resistance. We have shown in our earlier studies that CEBPD promotes inflammatory signaling and hypoxia adaptation by inhibiting the expression of the F-box protein FBXW7, a bona fide tumor suppressor (Balamurugan et al., EMBO J 2010; Nature Communications 2013). Because FBXW7 can target the stem cell factor Notch for degradation, we hypothesized that CEBPD can promote cell stemness. We tested this hypothesis using a variety of established breast and glioblastoma tumor cell lines/xenografts, primary mouse mammary tumor cells and tissues, recent patient-derived glioblastoma cell lines, as well as embryonic stem/cancer cell lines. Deletion or depletion of CEBPD reduced the number of stem cells as determined by surface markers, sphere formation, limiting dilution xenografts, and by expression of stemness genes. Mechanistic studies in culture models show that CEBPD is expressed in CSCs and connects and amplifies both hypoxia (HIF-1) and inflammation (IL-6/STAT3) pathways to increase Notch1 activity and cell stemness. Furthermore, we found that CEBPD also directly activates the expression of stemness associated genes such as OCT4, Sox2, KLF4, Myc, Nanog, CD44 and CD133. Taken together, this study provides novel insights into the molecular mechanisms that promote cancer cell stemness, and identifies a central role for CEBPD in integrating HIF-1α, IL-6/STAT3, and Notch1 signaling, while also activating expression of stemness factors. These results strongly suggest that pharmacological inhibition of CEBPD signaling may effectively target CSCs. Citation Format: Balamurugan Kuppusamy, Daniel Mendoza-Villaneuva, Glenn Summers, Shikha Sharan, Esta Sterneck. The CEBPD transcription factor: A signaling hub for promotion of cancer cell stemness. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A17.

Mitochondrial DNA Repair through OGG1 Activity Attenuates Breast Cancer Progression and Metastasis

Production of mitochondrial reactive oxygen species and integrity of mitochondrial DNA (mtDNA) are crucial in breast cancer progression and metastasis. Therefore, we evaluated the role of mtDNA damage in breast cancer by genetically modulating the DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1) in the PyMT transgenic mouse model of mammary tumorigenesis. We generated mice lacking OGG1 (KO), mice overexpressing human OGG1 subunit 1α in mitochondria (Tg), and mice simultaneously lacking OGG1 and overexpressing human OGG1 subunit 1α in mitochondria (KO/Tg). We found that Tg and KO/Tg mice developed significantly smaller tumors than KO and wild-type (WT) mice after 16 weeks. Histologic analysis revealed a roughly 2-fold decrease in the incidence of lung metastases in Tg mice (33.3%) compared to WT mice (62.5%). Furthermore, lungs from Tg mice exhibited nearly a 15-fold decrease in the average number of metastatic foci compared with WT mice (P ≤ 0.05). Primary tumors isolated from Tg mice also demonstrated reduced total and mitochondrial oxidative stress, diminished mtDNA damage, and increased mitochondrial function. Targeting hOGG1 to the mitochondria protected cells from mtDNA damage, resulting in downregulation of HIF1α and attenuated phosphorylation of Akt. Collectively, we demonstrate proof of concept that mtDNA damage results in breast cancer progression and metastasis in vivo. Moreover, our findings offer new therapeutic strategies for modulating the levels of mtDNA repair enzymes to delay or stall metastatic progression.

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

B7-H4 expression by nonhematopoietic cells in the tumor microenvironment promotes antitumor immunity.

The B7 family plays a critical role in both positive and negative regulation of immune responses by engaging a variety of receptors on lymphocytes. Importantly, blocking coinhibitory molecules using antibodies specific for CTLA-4 and PD-1 enhances tumor immunity in a subset of patients. Therefore, it is critical to understand the role of different B7 family members since they may be suitable therapeutic targets. B7-H4 is another member that inhibits T-cell function, and it is also upregulated on a variety of tumors and has been proposed to promote tumor growth. Here, we investigate the role of B7-H4 in tumor development and show that B7-H4 expression inhibits tumor growth in two mouse models. Furthermore, we show that B7-H4 expression is required for antitumor immune responses in a mouse model of mammary tumorigenesis. We found that the expression levels of B7-H4 correlate with MHC class I expression in both mouse and human samples. We show that IFNγ upregulates B7-H4 expression on mouse embryo fibroblasts and that the upregulation of B7-H4 on tumors is dependent on T cells. Notably, patients with breast cancer with increased B7-H4 expression show a prolonged time to recurrence. These studies demonstrate a positive role for B7-H4 in promoting antitumor immunity.

G protein-coupled estrogen receptor regulates mammary tumorigenesis and metastasis.

The role of 17β-estradiol (E2) in breast cancer development and tumor growth has traditionally been attributed exclusively to the activation of estrogen receptor-α (ERα). Although targeted inhibition of ERα is a successful approach for patients with ERα(+) breast cancer, many patients fail to respond or become resistant to anti-estrogen therapy. The discovery of the G protein-coupled estrogen receptor (GPER) suggested an additional mechanism through which E2 could exert its effects in breast cancer. Studies have demonstrated clinical correlations between GPER expression in human breast tumor specimens and increased tumor size, distant metastasis, and recurrence, as well as established a proliferative role for GPER in vitro; however, direct in vivo evidence has been lacking. To this end, a GPER-null mutation [GPER knockout (KO)] was introduced, through interbreeding, into a widely used transgenic mouse model of mammary tumorigenesis [MMTV-PyMT (PyMT)]. Early tumor development, assessed by the extent of hyperplasia and proliferation, was not different between GPER wild-type/PyMT (WT/PyMT) and those mice harboring the GPER-null mutation (KO/PyMT). However, by 12 to 13 weeks of age, tumors from KO/PyMT mice were smaller with decreased proliferation compared with those from WT/PyMT mice. Furthermore, tumors from the KO/PyMT mice were of histologically lower grade compared with tumors from their WT counterparts, suggesting less aggressive tumors in the KO/PyMT mice. Finally, KO/PyMT mice displayed dramatically fewer lung metastases compared with WT/PyMT mice. Combined, these data provide the first in vivo evidence that GPER plays a critical role in breast tumor growth and distant metastasis. This is the first description of a role for the novel estrogen receptor GPER in breast tumorigenesis and metastasis, demonstrating that it represents a new target in breast cancer diagnosis, prognosis, and therapy.

The serine protease inhibitor elafin maintains normal growth control by opposing the mitogenic effects of neutrophil elastase.

The serine protease inhibitor, elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared to normal human mammary epithelial cells (HMECs). Here, we evaluated the role of elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor deprived HMECs as they enter a quiescent (G0) state, suggesting that elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of elafin compromises the ability of HMECs to maintain G0-arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type elafin, but not elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of elafin to arrest cells in G0. In fact when purified NE was added to elafin knockdown HMECs, these cells demonstrated greater sensitivity to the growth promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples, and immunohistochemical analysis of normal breast tissue revealed robust elafin expression in the mammary epithelium; however, elafin expression was dramatically downregulated in a significant proportion of human breast tumor specimens. The loss of elafin expression during breast cancer progression may promote tumor growth as a consequence of increased NE-activity. To address the role of NE in mammary tumorigenesis, we next examined if deregulated NE-activity enhances mammary tumor growth. NE knockout in the C3(1)TAg mouse model of mammary tumorigenesis suppressed proliferation and reduced the kinetics of tumor growth. Overall, the imbalance between NE and its inhibitors, such as elafin, presents an important therapeutic target in breast cancer.

Force engages vinculin and promotes tumor progression by enhancing PI3K activation of phosphatidylinositol (3,4,5)-triphosphate.

Extracellular matrix (ECM) stiffness induces focal adhesion assembly to drive malignant transformation and tumor metastasis. Nevertheless, how force alters focal adhesions to promote tumor progression remains unclear. Here, we explored the role of the focal adhesion protein vinculin, a force-activated mechanotransducer, in mammary epithelial tissue transformation and invasion. We found that ECM stiffness stabilizes the assembly of a vinculin-talin-actin scaffolding complex that facilitates PI3K-mediated phosphatidylinositol (3,4,5)-triphosphate phosphorylation. Using defined two- and three-dimensional matrices, a mouse model of mammary tumorigenesis with vinculin mutants, and a novel super resolution imaging approach, we established that ECM stiffness, per se, promotes the malignant progression of a mammary epithelium by activating and stabilizing vinculin and enhancing Akt signaling at focal adhesions. Our studies also revealed that vinculin strongly colocalizes with activated Akt at the invasive border of human breast tumors, where the ECM is stiffest, and we detected elevated mechanosignaling. Thus, ECM stiffness could induce tumor progression by promoting the assembly of signaling scaffolds, a conclusion underscored by the significant association we observed between highly expressed focal adhesion plaque proteins and malignant transformation across multiple types of solid cancer. See all articles in this Cancer Research section, "Physics in Cancer Research."

Abstract C21: A germline polymorphism in Necdin causes changes in gene expression and mammary tumor metastasis

Abstract Necdin (NDN) is a transcription factor that regulates gene expression by cooperating with chromatin modifying factors. Earlier studies using mouse models of mammary tumorigenesis identified Ndn as a metastasis efficiency modifier that exerts its influence at the germline level. Differential expression of Ndn induces a gene expression signature that accurately predicts prognosis in human breast cancer. Mouse Ndn contains a non-synonymous germline single nucleotide polymorphism (SNP) (T50C; V17A) that distinguishes mouse strains with differing susceptibilities to mammary tumor metastasis. The aim of this study is to characterize how germline variation in Ndn regulates metastasis-associated transcription to enhance our understanding of how hereditary factors influence metastasis in breast cancer. We hypothesize that NDN, a known transcription factor, induces metastasis-predictive gene expression by interacting with chromatin to regulate transcription, and that Ndn coding variants differ in their capacity in regulating the transcription of target genes. To confirm the role of NDN in human breast cancer, haplotype analysis was performed in a population cohort consisting of 956 breast cancer patients. These analyses revealed that a two marker SNP haplotype in linkage disequilibrium with NDN is significantly associated with survival (P=0.0002). We examined the role of NDN in mammary tumor metastasis and transcriptional regulation by using cells stably over-expressing either the wildtype 17V or the variant 17A Ndn alleles in the highly metastatic Mvt-1 and 4T1 mouse mammary tumor cell lines. Orthotopic implantation of these cells into the mammary fat pad of NU/J and BALB/cJ mice demonstrated that cells expressing the wildtype Ndn 17V allele exhibit a significant decrease in pulmonary metastases compared to cells transduced with an empty vector control. However, these metastasis suppressive properties of Ndn were consistently lost in cells ectopically expressing the variant Ndn 17A allele, demonstrating a prominent role for this SNP in regulation of metastasis efficiency. Soft agar assays confirmed these results in vitro as well as, where a two-fold decrease in cell invasiveness was observed with the wildtype Ndn 17V allele compared to the control and cells over-expressing the variant Ndn 17A allele. Furthermore, microarray analyses revealed that a 71-gene expression signature accurately distinguished cells expressing the two Ndn allelic variants demonstrating that this SNP plays a significant part in gene regulation as well. These data collectively demonstrate that Ndn functions as a germline metastasis modifier, most likely by regulating gene expression at the transcriptional level. Additionally, our work illustrates that the Ndn V17A SNP plays a significant role in both metastasis and transcriptional regulation. Our ongoing work is focused on defining the mechanism of action of Ndn V17A SNP using ChIP-seq to identify target genes and pathways responsible for the profound influence of Ndn allelic variants on metastatic efficiency. Citation Format: Minnkyong Lee, Sarah M. Beggs, Jens Lichtenberg, Sujata B. Bupp, Niraj S. Trivedi, Ying Hu, David M. Bodine, Nigel P.S. Crawford. A germline polymorphism in Necdin causes changes in gene expression and mammary tumor metastasis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C21.

CDC25A Protein Stability Represents a Previously Unrecognized Target of HER2 Signaling in Human Breast Cancer: Implication for a Potential Clinical Relevance in Trastuzumab Treatment

The CDC25A-CDK2 pathway has been proposed as critical for the oncogenic action of human epidermal growth factor receptor 2 (HER2) in mammary epithelial cells. In particular, transgenic expression of CDC25A cooperates with HER2 in promoting mammary tumors, whereas CDC25A hemizygous loss attenuates the HER2-induced tumorigenesis penetrance. On the basis of this evidence of a synergism between HER2 and the cell cycle regulator CDC25A in a mouse model of mammary tumorigenesis, we investigated the role of CDC25A in human HER2-positive breast cancer and its possible implications in therapeutic response. HER2 status and CDC25A expression were assessed in 313 breast cancer patients and we found statistically significant correlation between HER2 and CDC25A (P = .007). Moreover, an HER2-positive breast cancer subgroup with high levels of CDC25A and very aggressive phenotype was identified (P = .005). Importantly, our in vitro studies on breast cancer cell lines showed that the HER2 inhibitor efficacy on cell growth and viability relied also on CDC25A expression and that such inhibition induces CDC25A down-regulation through phosphatidylinositol 3-kinase/protein kinase B pathway and DNA damage response activation. In line with this observation, we found a statistical significant association between CDC25A overexpression and trastuzumab-combined therapy response rate in two different HER2-positive cohorts of trastuzumab-treated patients in either metastatic or neoadjuvant setting (P = .018 for the metastatic cohort and P = .021 for the neoadjuvant cohort). Our findings highlight a link between HER2 and CDC25A that positively modulates HER2- targeted therapy response, suggesting that, in HER2-positive breast cancer patients, CDC25A overexpression affects trastuzumab sensitivity.

Abstract 3955: The tumor suppressor C/EBPdelta promotes hypoxia-induced EMT and expression of stem cell markers through inhibition of FBXW7: Implications for mammary tumor metastasis.

Abstract The transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) exhibits tumor-suppressor like-qualities such as downregulation of cyclin D1, and promotion of growth arrest and apoptosis. However, using a mouse model of mammary tumorigenesis (MMTV-Neu), we had found that C/EBPδ has dual functions. C/EBPδ suppresses primary tumor incidence while also augmenting metastatic progression. The pro-metastatic function of C/EBPδ can be correlated with a role in hypoxia adaptation and expression of pro-inflammatory genes and macrophage activation. C/EBPδ exerts both of these functions through inhibition of FBXW7, a component of SCF E3 ubiquitin ligase complex, which targets substrates such as c-myc and Notch for degradation (Balamurugan et al, 2010; Balamurugan et al., under revision). Hypoxia is believed to promote tumor progression in part by inducing epithelial-mesenchymal transition (EMT), which contributes to the generation of stem-like cancer cells (CSCs). In the present study, we found that hypoxia-induced EMT and expression of CSCs markers were compromised in Cebpd null primary tumor cells and in human breast tumor cells when C/EBPδ expression was silenced. On the other hand, silencing of the C/EBPδ-repressed gene FBXW7 was sufficient to induce a mesenchymal-like phenotype. Loss of FBXW7 has been shown to promote acquisition of stemness. In line with this notion, we found that overexpression of FBXW7 in the breast tumor cell line MDA-MB-468 reduced expression of aldehyde dehydrogenase 1 (Aldh1), C/EBPδ and the active form of NOTCH (NICD). Together, these data suggest that C/EBPδ augments - whereas its target FBXW7 prevents - EMT and breast cancer stem cell development, which may be an additional mechanism by which C/EBPδ promotes metastatic progression. Our current investigations are aimed at understanding how the different functions of C/EBPδ are integrated depending on cellular environment and context that results in its tumor promoting function despite its tumor suppressor role. Citation Format: Balamurugan Kuppusamy, Esta Sterneck. The tumor suppressor C/EBPdelta promotes hypoxia-induced EMT and expression of stem cell markers through inhibition of FBXW7: Implications for mammary tumor metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3955. doi:10.1158/1538-7445.AM2013-3955

The Many Faces of C/EBPδ and their Relevance for Inflammation and Cancer

The CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ) is a transcription factor that modulates many biological processes including cell differentiation, motility, growth arrest, proliferation, and cell death. The diversity of C/EBPδ's functions depends in part on the cell type and cellular context and can have opposing outcomes. For example, C/EBPδ promotes inflammatory signaling, but it can also inhibit pro-inflammatory pathways, and in a mouse model of mammary tumorigenesis, C/EBPδ reduces tumor incidence but promotes tumor metastasis. This review highlights the multifaceted nature of C/EBPδ's functions, with an emphasis on pathways that are relevant for cancer and inflammation, and illustrates how C/EBPδ emerged from the shadow of its family members as a fascinating “jack of all trades.” Our current knowledge on C/EBPδ indicates that, rather than being essential for a specific cellular process, C/EBPδ helps to interpret a variety of cues in a cell-type and context-dependent manner, to adjust cellular functions to specific situations. Therefore, insights into the roles and mechanisms of C/EBPδ signaling can lead to a better understanding of how the integration of different signaling pathways dictates normal and pathological cell functions and physiology.

Abstract SY10-04: Delineating cellular targets of transformation in breast cancer

Abstract To understand relationships between the mammary epithelia, ‘cells of origin’ and cancer stem cells in breast tumors, it is necessary to dissect the normal mammary epithelial hierarchy. We have isolated discrete populations of mouse and human mammary epithelial cells on the basis of cell-surface markers and identified cell subsets that are highly enriched for mammary stem (MaSC), luminal progenitor and mature luminal cells. Analysis of different mouse models of mammary tumorigenesis using syngeneic transplantation assays and interrogation of molecular profiles has revealed potential ‘cells of origin’ in preneoplastic tissue and the presence of a definitive cancer stem cell subset in different mouse mammary tumors. Cell lineage tracing studies in the mouse provide the current gold standard for identifying cells of origin. Towards this end, we have constructed a series of transgenic mice harboring lineage-specific gene regulatory regions (180 kB) that can be used to direct the expression of specific mammary oncogenes to discrete epithelial cell types. This will allow us to address the contribution of ‘cells of origin’ and the nature of the oncogene itself to mouse mammary tumorigenesis. 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 SY10-04. doi:1538-7445.AM2012-SY10-04

β1-integrins signaling and mammary tumor progression in transgenic mouse models: implications for human breast cancer

Consistent with their essential role in cell adhesion to the extracellular matrix, integrins and their associated signaling pathways have been shown to be involved in cell proliferation, migration, invasion and survival, processes required in both tumorigenesis and metastasis. β1-integrins represent the predominantly expressed integrins in mammary epithelial cells and have been proven crucial for mammary gland development and differentiation. Here we provide an overview of the studies that have used transgenic mouse models of mammary tumorigenesis to establish β1-integrin as a critical mediator of breast cancer progression and thereby as a potential therapeutic target for the development of new anticancer strategies.

Abstract 117: The role of microRNAs in mammary tumorigenesis

Abstract MicroRNAs (miRNAs) are small non-coding regulatory RNA molecules that act post-transcriptionally on mRNA to regulate the expression of most cellular genes. Many miRNAs are aberrantly expressed in tumor tissue, acting either as tumor suppressors or oncogenes, and their expression has been implicated in the development of breast cancer. Using a transgenic mouse model of mammary tumorigenesis where transgenic overexpression of IGF-IR drives mammary tumor development (MTB-IGFIR transgenic mice), a miRNA array was performed to study the expression level of various miRNAs in mammary tumors compared to wild-type mammary tissue. The miRNA array revealed that 43 miRNAs were significantly elevated by a minimum of 2-fold in tumor tissue, while 45 miRNAs were significantly downregulated by at least 2-fold in tumor tissue when compared to normal mammary tissue. The expression differences of miRNAs (miR-24, miR-31, miR-183, miR-200a*, miR-200b, miR-200c, miR-210, miR-378 and miR-574-3p) were confirmed in tumor and normal mammary tissues using qRT-PCR. These miRNAs were chosen as they displayed some of the largest expression differences between the mammary tumors and normal mammary tissue, and several have been implicated in tumor progression and metastasis. In particular, miR-200c and miR-31 levels are inversely related to metastasis, whereas miR-183 may be involved in reducing cellular migratory properties. Next, the expression of these miRNAs was examined in a tumor cell line (RM11A) generated from a MTB-IGFIR mammary tumor. We found that miR-31, miR-183, miR-200c, miR-210, and miR-378 were expressed in the RM11A cells. We then used synthetic miRNA precursors that mimic the mature form of miRNAs and inhibitors to overexpress and knockdown, respectively, the levels of these miRNAs in RM11A cells. Overexpression of these five miRNAs was optimized using qRT-PCR with TaqMan primers to increase expression levels between 30 and 400 fold, as this magnitude of change was observed in the miRNA array. To determine the impact of miRNAs on mammary tumorigenesis, assays for proliferation (Ki-67 immunofluorescence), migration (scratch wound and Boyden chamber), and cell survival (JC-1 fluorescence) will be performed. To date we have observed that when compared to negative controls, overexpression of miR-210 significantly increased proliferation by approximately 1.5- fold, while the overexpression of miR-31, miR-183, and miR-378 did not significantly affect proliferation. Functional assays performed in the RM11A cell line will be repeated in a second mammary tumour cell line (4T1) to compare and contrast the importance of these miRNAs in another cell line. Discovering the differential expression of miRNAs and their effects on proliferation, migration, and survival in the RM11A and 4T1 cells will enhance our understanding of the function of miRNAs in human 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 117. doi:10.1158/1538-7445.AM2011-117

Abstract SY12-03: Getting abreast of the mammary epithelial hierarchy and breast cancer

Abstract To understand relationships between breast tissue, cells of origin and cancer stem cells in breast tumors, it is necessary to dissect the normal mammary epithelial hierarchy. We have isolated discrete populations of mouse and human mammary epithelial cells on the basis of cell-surface markers and defined subpopulations that are highly enriched for mammary stem, luminal progenitor and mature luminal cells. Transcriptome analyses of mouse and human mammary epithelial populations has led to the identification of multiple conserved genes and pathways. Analysis of different mouse models of mammary tumorigenesis using syngeneic transplantation assays and interrogation of molecular profiles has revealed potential cells of origin in preneoplastic tissue and the presence of a definitive cancer stem cell subset in different mouse mammary tumors. The role of the zinc finger transcription factor Gata-3, a critical regulator of luminal cell differentiation and a favourable prognostic indicator in breast cancer, has recently been explored in tumor initiation using mouse models. Gata-3 deficiency was found to increase the tumorigenicity of different epithelial subpopulations, with accompanying cell-fate changes. In the context of human breast tissue, analogous studies have enabled us to pinpoint the cellular defect occurring in precancerous tissue from BRCA1 mutation carriers. These carriers were shown to harbor aberrant luminal progenitor cells. Combined with interrogation of the molecular profiles of the different subtypes of breast cancer, the luminal progenitor was identified as the likely cell of origin for BRCA1-associated basal cancers. 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 SY12-03. doi:10.1158/1538-7445.AM2011-SY12-03

Abstract P4-04-03: Transcriptome Analyses of Mouse and Human Mammary Cell Subpopulations Reveals Multiple Conserved Genes and Pathways

Abstract INTRODUCTION: Molecular characterization of the normal epithelial cell types that reside in the mammary gland is an important step toward understanding pathways that regulate self-renewal, lineage commitment, and differentiation along the hierarchy. We previously reported distinct stages in human mammary epithelial cell development and linked them to breast tumor subtypes previously defined by gene expression profiling. Here we determined the gene expression signatures of four distinct subpopulations isolated from the mouse mammary gland. The epithelial cell signatures were used to interrogate mouse models of mammary tumorigenesis and to compare with their normal human counterpart subsets to identify conserved genes and networks. METHODS: RNA was prepared from freshly sorted mouse mammary cell subpopulations (mammary stem cell (MaSC)-enriched, committed luminal progenitor, mature luminal and stromal cell) and used for gene expression profiling analysis on the Illumina platform. Gene signatures were derived and compared with those previously reported for the analogous normal human mammary cell subpopulations. The mouse and human epithelial subset signatures were then subjected to Ingenuity Pathway Analysis (IPA) to identify conserved pathways. RESULTS: The four mouse mammary cell subpopulations exhibited distinct gene signatures. Comparison of these signatures with the molecular profiles of different mouse models of mammary tumorigenesis revealed that tumors arising in MMTV-Wnt-1 and p53-/- mice were enriched for MaSC-subset genes, whereas the gene profiles of MMTV-Neu and MMTV-PyMT tumors were most concordant with the luminal progenitor cell signature. Comparison of the mouse mammary epithelial cell signatures with their human counterparts revealed substantial conservation of genes, whereas IPA highlighted a number of conserved pathways in the three epithelial subsets. CONCLUSIONS: The conservation of genes and pathways across species further validates the use of the mouse as a model to study mammary gland development and highlights pathways that are likely to govern cell-fate decisions and differentiation. It is noteworthy that many of the conserved genes in the MaSC population have been considered as epithelialmesenchymal transition (EMT) signature genes. Therefore, the expression of these genes in tumor cells may reflect basal epithelial cell characteristics and not necessarily cells that have undergone an EMT. Comparative analyses of normal mouse epithelial subsets with murine tumor models have implicated distinct cell types in contributing to tumorigenesis in the different models. Figures available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-04-03.

Insights into the cell of origin in breast cancer and breast cancer stem cells

The precise cell types that give rise to tumors and mechanisms that underpin tumor heterogeneity are poorly understood. There is increasing evidence to suggest that diverse solid tumors are hierarchically organized and may be sustained by a distinct subpopulation of cancer stem cells (CSCs). The CSC hypothesis provides an attractive cellular mechanism that can account for the therapeutic refractoriness and dormant behavior exhibited by many tumor types. Breast cancer was the first solid malignancy from which CSCs were identified and isolated. Direct evidence for the CSC hypothesis has also recently emerged from mouse models of mammary tumorigenesis, although alternative models to explain heterogeneity also seem to apply. Our group has found that the luminal epithelial progenitor marker CD61/beta3 integrin identified a CSC population in mammary tumors from MMTV-wnt-1 mice. However, no CSCs could be identified in the more homogeneous MMTV-neu/erbB2 model, suggesting an alternate (clonal evolution or stochastic) model of tumorigenesis. It seems likely that both paradigms of tumor propagation exist in human cancer. From a clinical perspective, the CSC concept has significant implications. Quiescent CSCs are thought to be more resistant to chemotherapy and targeted therapy. Enrichment of putative CSCs has been noted in studies of chemotherapy-treated patients, lending support to the CSC hypothesis and their potential role in chemoresistance. Although many unresolved questions on CSCs remain, ongoing efforts to identify and characterize CSCs continue to be an important area of investigation, with the potential to identify novel tumor targeting strategies.

Abstract 2372: The metastasis efficiency modifier ribosomal RNA processing 1 homologue (RRP1B) is a chromatin-associated factor

Abstract Previous experiments using the polyoma middle T (PyMT) mouse model of mammary tumorigenesis have demonstrated that host germline variation modulates metastasis in breast cancer. Recent work using the PyMT model has identified novel metastasis susceptibility genes, an example of which includes Rrp1b (ribosomal RNA processing 1 homolog B). Over-expression of Rrp1b in a mouse mammary tumor cell line induces a gene expression signature that predicts survival in a well characterized Dutch breast cancer dataset. Additionally, a polymorphism was identified within human RRP1B (G1421A; P436L), which is associated with differential survival in multiple breast cancer cohorts. Here we extend the analysis of RRP1B function by demonstrating that the Rrp1b activation gene expression signature accurately predicts survival in three additional publicly available breast carcinoma gene expression datasets. We have also investigated the mechanism of action of RRP1B by defining protein binding partners for this germline-encoded metastasis modifier. Tandem affinity purification demonstrated that RRP1B physically interacts with many nucleosome binding factors, all of which have been implicated in regulation of gene expression. Examples include the linker histone H1X, poly(ADP-ribose) polymerase 1, tripartite motif-containing 28 (TIF-1β/KAP1), and cold shock domain protein A. These interactions were confirmed by co-immunofluorescence (Co-IF) and co-immunoprecipitation (Co-IP). Additionally, co-IF experiments demonstrated that RRP1B is localized to the perinuclear and perinucleolar regions, which is a particularly interesting given that factors localized to these regions are often involved in heterochromatinization and gene silencing. Furthermore, co-IP and co-IF demonstrated that RRP1B interacts with heterochromatin protein-1α and acetyl-histone H4 lysine 5, suggesting that RRP1B may well regulate gene expression through interactions with heterochromatin and euchromatin. Finally, we investigated the effects of ectopic expression of the 436Pro and 436Leu RRP1B allelic variants that have previously been associated with differential survival in breast cancer. Gene expression analyses demonstrate that, compared with ectopic expression of wildtype 436 Pro RRP1B in HeLa cells, the variant 436Leu RRP1B differentially modulates various transcription factors controlled by TRIM28 and CSDA. These data suggest that RRP1B, a tumor progression and metastasis susceptibility candidate gene, is potentially a dynamic modulator of transcription and chromatin structure. 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 2372.