The comprehensive role of E-cadherin in maintaining prostatic epithelial integrity during oncogenic transformation and tumor progression.

E-cadherin complexes with the actin cytoskeleton via cytoplasmic catenins and maintains the functional characteristics and integrity of the epithelia in normal epithelial tissues. Lost expression of E-cadherin disrupts this complex resulting in loss of cell polarity, epithelial denudation and increased epithelial permeability in a variety of tissues. Decreased expression of E-cadherin has also been observed in invasive and metastatic human tumors. In this study, we investigated the effect of E-cadherin loss in prostatic epithelium using newly developed genetically engineered mouse models. Deletion of E-cadherin in prostatic luminal epithelial cells with modified probasin promoter driven Cre (PB-Cre4) induced the development of mouse prostatic intraepithelial neoplasia (PIN). An increase in levels of cytoplasmic and nuclear ß-catenin appeared in E-cadherin deleted atypical cells within PIN lesions. Using various experimental approaches, we further demonstrated that the knockdown of E-cadherin expression elevated free cytoplasmic and nuclear ß-catenin and enhanced androgen-induced transcription and cell growth. Intriguingly, pathological changes representing prostatic epithelial cell denudation and increased apoptosis accompanied the above PIN lesions. The essential role of E-cadherin in maintaining prostatic epithelial integrity and organization was further demonstrated using organoid culture approaches. To directly assess the role of loss of E-cadherin in prostate tumor progression, we generated a new mouse model with bigenic Cdh1 and Pten deletion in prostate epithelium. Early onset, aggressive tumor phenotypes presented in the compound mice. Strikingly, goblet cell metaplasia was observed, intermixed within prostatic tumor lesions of the compound mice. This study provides multiple lines of novel evidence demonstrating a comprehensive role of E-cadherin in maintaining epithelial integrity during the course of prostate oncogenic transformation, tumor initiation and progression.

PIK3CA(H1047R) induces multipotency and multi-lineage mammary tumours.

The adult mouse mammary epithelium contains self-sustained cell lineages that form the inner luminal and outer basal cell layers, with stem and progenitor cells contributing to its proliferative and regenerative potential. A key issue in breast cancer biology is the effect of genomic lesions in specific mammary cell lineages on tumour heterogeneity and progression. The impact of transforming events on fate conversion in cancer cells of origin and thus their contribution to tumour heterogeneity remains largely elusive. Using in situ genetic lineage tracing and limiting dilution transplantation, we have unravelled the potential of PIK3CA(H1047R), one of the most frequent mutations occurring in human breast cancer, to induce multipotency during tumorigenesis in the mammary gland. Here we show that expression of PIK3CA(H1047R) in lineage-committed basal Lgr5-positive and luminal keratin-8-positive cells of the adult mouse mammary gland evokes cell dedifferentiation into a multipotent stem-like state, suggesting this to be a mechanism involved in the formation of heterogeneous, multi-lineage mammary tumours. Moreover, we show that the tumour cell of origin influences the frequency of malignant mammary tumours. Our results define a key effect of PIK3CA(H1047R) on mammary cell fate in the pre-neoplastic mammary gland and show that the cell of origin of PIK3CA(H1047R) tumours dictates their malignancy, thus revealing a mechanism underlying tumour heterogeneity and aggressiveness.

ß-Catenin haploinsufficiency promotes mammary tumorigenesis in an ErbB2-positive basal breast cancer model.

Aberrant activation of ß-catenin through its activity as a transcription factor has been observed in a large proportion of human malignancies. Despite the improved understanding of the ß-catenin signaling pathway over the past three decades, attempts to develop therapies targeting ß-catenin remain challenging, and none of these targeted therapies have advanced to the clinic. In this study, we show that part of the challenge in antagonizing ß-catenin is caused by its dual functionality as a cell adhesion molecule and a signaling molecule. In a mouse model of basal ErbB2 receptor tyrosine kinase 2 (ErbB2)-positive breast cancer (ErbB2KI), which exhibits aberrant ß-catenin nuclear signaling, ß-catenin haploinsufficiency induced aggressive tumor formation and metastasis by promoting the disruption of adherens junctions, dedifferentiation, and an epithelial to mesenchymal transition (EMT) transcriptional program. In contrast to the accelerated tumor onset observed in the haploid-insufficient ErbB2 tumors, deletion of both ß-catenin alleles in the ErbB2KI model had only a minor impact on tumor onset that further correlated with the retention of normal adherens junctions. We further showed that retention of adherens junctional integrity was caused by the up-regulation of the closely related family member plakoglobin (γ-catenin) that maintained both adherens junctions and the activation of Wnt target genes. In contrast to the ErbB2KI basal tumor model, modulation of ß-catenin levels had no appreciable impact on tumor onset in an ErbB2-driven model of luminal breast cancer [murine mammary tumor virus promoter (MMTV-NIC)]. These observations argue that the balance of junctional and nuclear ß-catenin activity has a profound impact on tumor progression in this basal model of ErbB2-positive breast cancer.

The atypical cyclin-like protein Spy1 overrides p53-mediated tumour suppression and promotes susceptibility to breast tumourigenesis.

BACKGROUND: Breast cancer is the most common cancer to affect women and one of the leading causes of cancer-related deaths. Proper regulation of cell cycle checkpoints plays a critical role in preventing the accumulation of deleterious mutations. Perturbations in the expression or activity of mediators of cell cycle progression or checkpoint activation represent important events that may increase susceptibility to the onset of carcinogenesis. The atypical cyclin-like protein Spy1 was isolated in a screen for novel genes that could bypass the DNA damage response. Clinical data demonstrates that protein levels of Spy1 are significantly elevated in ductal and lobular carcinoma of the breast. We hypothesized that elevated Spy1 would override protective cell cycle checkpoints and support the onset of mammary tumourigenesis. METHODS: We generated a transgenic mouse model driving expression of Spy1 in the mammary epithelium. Mammary development, growth characteristics and susceptibility to tumourigenesis were studied. In vitro studies were conducted to investigate the relationship between Spy1 and p53. RESULTS: We found that in the presence of wild-type p53, Spy1 protein is held 'in check' via protein degradation, representing a novel endogenous mechanism to ensure protected checkpoint control. Regulation of Spy1 by p53 is at the protein level and is mediated in part by Nedd4. Mutation or abrogation of p53 is sufficient to allow for accumulation of Spy1 levels resulting in mammary hyperplasia. Sustained elevation of Spy1 results in elevated proliferation of the mammary gland and susceptibility to tumourigenesis. CONCLUSIONS: This mouse model demonstrates for the first time that degradation of the cyclin-like protein Spy1 is an essential component of p53-mediated tumour suppression. Targeting cyclin-like protein activity may therefore represent a mechanism of re-sensitizing cells to important cell cycle checkpoints in a therapeutic setting.

Pathobiology of the 129:Stat1 -/- mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype.

BACKGROUND: Stat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1 tm1Rds) develop estrogen receptor-positive (ER+), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1 -/- host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors. METHODS: Mice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1 -/--derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells. RESULTS: Tumorigenesis in 129:Stat1 -/- originates from a population of FoxA1+ large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1 -/- tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor. CONCLUSIONS: 129:Stat1 -/- is a unique model for studying the critical origins and risk reduction strategies in age-related ER+ breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies.

Mice deficient in Rbm38, a target of the p53 family, are susceptible to accelerated aging and spontaneous tumors.

RNA-binding motif protein 38 (Rbm38), also called RNPC1 [RNA-binding region (RNP1, RRM) containing 1], is a target of the p53 family and modulates p53 expression via mRNA translation. To investigate the biological function of Rbm38 in vivo, we generated an Rbm38-null mouse model. We showed that mice deficient in Rbm38 exhibit signs of accelerated aging and are prone to hematopoietic defects and spontaneous tumors. To determine the biological significance of the p53-Rbm38 loop, we showed that Rbm38 deficiency enhances accumulation of p53 induced by ionizing radiation (IR) and sensitizes mice to IR-induced lethality in a p53-dependent manner. Most importantly, Rbm38 deficiency markedly decreases the tumor penetrance in mice heterozygous for p53 via enhanced p53 expression. Interestingly, we found that Rbm38 deficiency shortens the life span of, and promotes lymphomagenesis in, mice deficient in p53. These results provide genetic evidence that Rbm38 is necessary for normal hematopoiesis and for suppressing accelerated aging and tumorigenesis. Thus, the p53-Rbm38 axis might be explored for extending longevity and for tumor suppression.

The Trp53 delta proline (Trp53ΔP) mouse exhibits increased genome instability and susceptibility to radiation-induced, but not spontaneous, tumor development.

The tumor suppressor TP53 can initiate a plethora of anti-proliferative effects to maintain genomic integrity under conditions of genotoxic stress. The N-terminal proline-rich domain (PRD) of TP53 is important in the regulation of TP53 activity and stability. A common polymorphism at codon 72 in this region has been associated with altered cancer risk in humans. The Trp53ΔP mouse, which carries a germline homozygous deletion of a region of the PRD, does not develop spontaneous tumors in a mixed 129/Sv and C57BL/6 genetic background, but is highly susceptible to a broad range of tumor types following total body exposure to 4 Gy gamma (γ) radiation. This contrasts with the tumor spectrum in Trp53 null (-/-) mice, which mainly develop thymic lymphomas and osteosarcomas. Analysis of genomic instability in tissues and cells from Trp53ΔP mice demonstrated elevated basal levels of aneuploidy, but this is not sufficient to drive spontaneous tumorigenesis, which requires an additional DNA damage stimulus. Levels of genomic instability did not increase significantly in Trp53ΔP mice following irradiation exposure, suggesting that other radiation effects including tissue inflammation, altered metabolism or autophagy, may play an important role. The Trp53ΔP mouse is a novel model to dissect the mechanisms of tumor development induced by radiation exposure. © 2015 Wiley Periodicals, Inc.

Insights from transgenic mouse models of ERBB2-induced breast cancer.

One-third of patients with breast cancer overexpress the ERBB2 receptor tyrosine kinase, which is associated not only with a more aggressive phenotype but also reduced responsiveness to hormonal therapies. Over the past two decades, many ERBB2 mouse models for breast cancer have conclusively shown that this receptor has a causal role in breast cancer development. These mouse models have also enabled the mechanisms controlling tumour growth, angiogenesis, metastasis, dormancy and recurrence in ERBB2-positive breast cancer to be elucidated. In addition, a mouse model has recently been described that accurately recapitulates many of the hallmarks associated with the early stages of the human disease.

ETV4 promotes metastasis in response to activation of PI3-kinase and Ras signaling in a mouse model of advanced prostate cancer.

Combinatorial activation of PI3-kinase and RAS signaling occurs frequently in advanced prostate cancer and is associated with adverse patient outcome. We now report that the oncogenic Ets variant 4 (Etv4) promotes prostate cancer metastasis in response to coactivation of PI3-kinase and Ras signaling pathways in a genetically engineered mouse model of highly penetrant, metastatic prostate cancer. Using an inducible Cre driver to simultaneously inactivate Pten while activating oncogenic Kras and a fluorescent reporter allele in the prostate epithelium, we performed lineage tracing in vivo to define the temporal and spatial occurrence of prostate tumors, disseminated tumor cells, and metastases. These analyses revealed that though disseminated tumors cells arise early following the initial occurrence of prostate tumors, there is a significant temporal lag in metastasis, which is temporally coincident with the up-regulation of Etv4 expression in primary tumors. Functional studies showed that knockdown of Etv4 in a metastatic cell line derived from the mouse model abrogates the metastatic phenotype but does not affect tumor growth. Notably, expression and activation of ETV4, but not other oncogenic ETS genes, is correlated with activation of both PI3-kinase and Ras signaling in human prostate tumors and metastases. Our findings indicate that ETV4 promotes metastasis in prostate tumors that have activation of PI3-kinase and Ras signaling, and therefore, ETV4 represents a potential target of therapeutic intervention for metastatic prostate cancer.

Met synergizes with p53 loss to induce mammary tumors that possess features of claudin-low breast cancer.

Triple-negative breast cancer (TNBC) accounts for ∼20% of cases and contributes to basal and claudin-low molecular subclasses of the disease. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53), and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC and transgenic Met models (Met(mt)) develop basal-like tumors. To investigate collaborating events in the genesis of TNBC, we generated Met(mt) mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Met(mt), significantly increased tumor penetrance over Met(mt) or Trp53 loss alone. Unlike Met(mt) tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Met(mt) tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins, an epithelial-to-mesenchymal transition signature, and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, in which MET inhibitors restore cell-cell junctions, rescue claudin 1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilized TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature, and provides a rationale for anti-MET therapies in TNBC.

Multipotent luminal mammary cancer stem cells model tumor heterogeneity.

INTRODUCTION: The diversity of human breast cancer subtypes has led to the hypothesis that breast cancer is actually a number of different diseases arising from cells at various stages of differentiation. The elusive nature of the cell(s) of origin thus hampers approaches to eradicate the disease. METHODS: Clonal cell lines were isolated from primary transgenic polyomavirus middle T (PyVmT) luminal tumors. Mammary cancer stem cell (MaCSC) properties were examined by immunofluorescence, flow cytometry, differentiation assays and in vivo tumorigenesis. RESULTS: Clonal cell lines isolated from primary PyVmT mouse mammary luminal tumors can differentiate into luminal, myoepithelial, alveolar and adipocyte lineages. Upon orthotopic injection, progeny of a single cell follow a pattern of progression from ductal carcinoma in situ, to adenoma, adenocarcinoma and epithelial metastasis that recapitulates the transgenic model. Tumors can evolve in vivo from hormone receptor-positive to hormone receptor-negative Her2-positive, or triple negative CD44hi basal-like and claudin-low tumors. Contrary to the current paradigm, we have defined a model in which multiple tumor subtypes can originate from a single multipotent cancer stem cell that undergoes genetic and/or epigenetic evolution during tumor progression. As in human tumors, the more aggressive tumor subtypes express nuclear p53. Tumor cell lines can also be derived from these more advanced tumor subtypes. CONCLUSIONS: Since the majority of human tumors are of the luminal subtype, understanding the cell of origin of these tumors and how they relate to other tumor subtypes will impact cancer therapy. Analysis of clonal cell lines derived from different tumor subtypes suggests a developmental hierarchy of MaCSCs, which may provide insights into the progression of human breast cancer.

Introduction of Zinc-salt Fixation for Effective Detection of Immune Cell-related Markers by Immunohistochemistry.

Tissue localization of immune cells is critical to the study of disease processes in mouse models of human diseases. However, immunohistochemistry (IHC) for immune cell phenotyping in mouse tissue sections presents specific technical challenges. For example, CD4 and CD8 have been difficult to detect using IHC on formalin-fixed and paraffin-embedded mouse tissue, prompting alternative methods. We investigated the use of formalin-free zinc-salt fixation (ZN) and optimized IHC protocols for detecting a panel of immune cell-related markers (CD3, CD4, CD8, Foxp3, B220, F4/80, CD68, and major histocompatibility complex [MHC] class-I, MHC class-II, and Gr-1). The IHC results for these markers were compared on mouse spleen tissue treated with neutral buffered formalin (NBF) or ZN with or ZN without antigen retrieval (AR). Whereas CD4 and CD8 were not detected in NBF-treated tissue, all markers were detected in ZN-treated tissue without AR. Thus, the use of ZN treatment for IHC staining can be a good tool for studying immunoreactive lesions in tissues.

Diet-induced metabolic change induces estrogen-independent allometric mammary growth.

Lifetime breast cancer risk reflects an unresolved combination of early life factors including diet, body mass index, metabolic syndrome, obesity, and age at first menses. In parallel, the onset of allometric growth by the mammary glands around puberty is widely held to be estrogen (E)-dependent. Here we report that several physiological changes associated with metabolic syndrome in response to a diet supplemented with the trans-10, cis-12 isomer of conjugated linoleic acid lead to ovary-independent allometric growth of the mammary ducts. The E-independence of this diet-induced growth was highlighted by the fact that it occurred both in male mice and with pharmacological inhibition of either E receptor function or E biosynthesis. Reversal of the metabolic phenotype with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone abrogated diet-induced mammary growth. A role for hyperinsulinemia and increased insulin-like growth factor-I receptor (IGF-IR) expression during mammary growth induced by the trans-10, cis-12 isomer of conjugated linoleic acid was confirmed by its reversal upon pharmacological inhibition of IGF-IR function. Diet-stimulated ductal growth also increased mammary tumorigenesis in ovariectomized polyomavirus middle T-antigen mice. Our data demonstrate that diet-induced metabolic dysregulation, independently of ovarian function, stimulates allometric growth within the mammary glands via an IGF-IR-dependent mechanism.

An integrin binding-defective mutant of insulin-like growth factor-1 (R36E/R37E IGF1) acts as a dominant-negative antagonist of the IGF1 receptor (IGF1R) and suppresses tumorigenesis but still binds to IGF1R.

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvß3 and α6ß4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.

Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis.

Metastatic disease is the primary cause of death in breast cancer, the most common malignancy in Western women. Loss of E-cadherin is associated with tumor metastasis, as well as with invasive lobular carcinoma (ILC), which accounts for 10%-15% of all breast cancers. To study the role of E-cadherin in breast oncogenesis, we have introduced conditional E-cadherin mutations into a mouse tumor model based on epithelium-specific knockout of p53. Combined loss of E-cadherin and p53 resulted in accelerated development of invasive and metastatic mammary carcinomas, which show strong resemblance to human ILC. Moreover, loss of E-cadherin induced anoikis resistance and facilitated angiogenesis, thus promoting metastatic disease. Our results suggest that loss of E-cadherin contributes to both mammary tumor initiation and metastasis.

Nonlinear progression across the occult transition establishes cancer lethality.

Cancer screening is based upon a linear model of growth and invasion. Yet, early dissemination during the lengthy pre-diagnostic phase suggests that nonlinearity in growth can also occur. Therefore, we quantitatively traced the invisible and visible phases of tumorigenesis in the mammary gland for more than two-thousand tumors. Dynamic mathematical models of the invisible phase revealed an occult checkpoint resulting in nonlinear progression of transformed field cells. We found that expansile fields have increased dwell time at the occult checkpoint resulting in a large reservoir of image detectable precursors prior to invasion. In contrast, slowly proliferating lesions disseminate early and then transition rapidly through an occult checkpoint in a process we term nascent lethality. Our data illustrate how nonlinear growth across an occult checkpoint can account for a paradoxical increase in early-stage cancer detection without a dramatic reduction in metastatic burden. Highlights: Growth during the invisible phase of tumorigenesis is a nonlinear processField size and field growth rate are uncoupled from metastatic potentialOccult transition rates vary by genotypeNascent lethal lesions are currently undetectable.

Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers.

Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER+/PR+ model (SSM2), a Her2+ model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters.

The transcriptional repressor Snail promotes mammary tumor recurrence.

Breast cancer recurrence is a fundamental clinical manifestation of tumor progression and represents the principal cause of death from this disease. Using a conditional transgenic mouse model for the recurrence of HER2/neu-induced mammary tumors, we demonstrate that the transcriptional repressor Snail is spontaneously upregulated in recurrent tumors in vivo and that recurrence is accompanied by epithelial-to-mesenchymal transition (EMT). Consistent with a causal role for Snail in these processes, we show that Snail is sufficient to induce EMT in primary tumor cells, that Snail is sufficient to promote mammary tumor recurrence in vivo, and that high levels of Snail predict decreased relapse-free survival in women with breast cancer. In aggregate, our observations strongly implicate Snail in the process of breast cancer recurrence.

Early vascular deficits are correlated with delayed mammary tumorigenesis in the MMTV-PyMT transgenic mouse following genetic ablation of the NG2 proteoglycan.

INTRODUCTION: The neuron-glial antigen 2 (NG2) proteoglycan promotes pericyte recruitment and mediates pericyte interaction with endothelial cells. In the absence of NG2, blood vessel development is negatively impacted in several pathological models. Our goal in this study was to determine the effect of NG2 ablation on the early development and function of blood vessels in mammary tumors in the mammary tumor virus-driven polyoma middle T (MMTV-PyMT) transgenic mouse, and to correlate these vascular changes with alterations in mammary tumor growth. METHODS: Three different tumor paradigms (spontaneous tumors, transplanted tumors, and orthotopic allografts of tumor cell lines) were used to investigate the effects of NG2 ablation on breast cancer progression in the MMTV-PyMT transgenic mouse. In addition to examining effects of NG2 ablation on mammary tumor growth, we also investigated effects on the structure and function of tumor vasculature. RESULTS: Ablation of NG2 led to reduced early progression of spontaneous, transplanted, and orthotopic allograft mammary tumors. NG2 was not expressed by the mammary tumor cells themselves, but instead was found on three components of the tumor stroma. Microvascular pericytes, myeloid cells, and adipocytes were NG2-positive in both mouse and human mammary tumor stroma. The effect of NG2 on tumor progression therefore must be stromal in nature. Ablation of NG2 had several negative effects on early development of the mammary tumor vasculature. In the absence of NG2, pericyte ensheathment of endothelial cells was reduced, along with reduced pericyte maturation, reduced sprouting of endothelial cells, reduced assembly of the vascular basal lamina, and reduced tumor vessel diameter. These early deficits in vessel structure are accompanied by increased vessel leakiness, increased tumor hypoxia, and decreased tumor growth. NG2 ablation also diminishes the number of tumor-associated and TEK tyrosine kinase endothelial (Tie2) expressing macrophages in mammary tumors, providing another possible mechanism for reducing tumor vascularization and growth. CONCLUSIONS: These results emphasize the importance of NG2 in mediating pericyte/endothelial cell communication that is required for proper vessel maturation and function. In the absence of normal pericyte/endothelial cell interaction, poor vascular function results in diminished early progression of mammary tumors.

STAT1-deficient mice spontaneously develop estrogen receptor α-positive luminal mammary carcinomas.

INTRODUCTION: Although breast cancers expressing estrogen receptor-α (ERα) and progesterone receptors (PR) are the most common form of mammary malignancy in humans, it has been difficult to develop a suitable mouse model showing similar steroid hormone responsiveness. STAT transcription factors play critical roles in mammary gland tumorigenesis, but the precise role of STAT1 remains unclear. Herein, we show that a subset of human breast cancers display reduced STAT1 expression and that mice lacking STAT1 surprisingly develop ERα+/PR+ mammary tumors. METHODS: We used a combination of approaches, including histological examination, gene targeted mice, gene expression analysis, tumor transplantaion, and immunophenotyping, to pursue this study. RESULTS: Forty-five percent (37/83) of human ERα+ and 22% (17/78) of ERα- breast cancers display undetectable or low levels of STAT1 expression in neoplastic cells. In contrast, STAT1 expression is elevated in epithelial cells of normal breast tissues adjacent to the malignant lesions, suggesting that STAT1 is selectively downregulated in the tumor cells during tumor progression. Interestingly, the expression levels of STAT1 in the tumor-infiltrating stromal cells remain elevated, indicating that single-cell resolution analysis of STAT1 level in primary breast cancer biopsies is necessary for accurate assessment. Female mice lacking functional STAT1 spontaneously develop mammary adenocarcinomas that comprise > 90% ERα+/PR+ tumor cells, and depend on estrogen for tumor engraftment and progression. Phenotypic marker analyses demonstrate that STAT1-/- mammary tumors arise from luminal epithelial cells, but not myoepithelial cells. In addition, the molecular signature of the STAT1-/- mammary tumors overlaps closely to that of human luminal breast cancers. Finally, introduction of wildtype STAT1, but not a STAT1 mutant lacking the critical Tyr701 residue, into STAT1-/- mammary tumor cells results in apoptosis, demonstrating that the tumor suppressor function of STAT1 is cell-autonomous and requires its transcriptional activity. CONCLUSIONS: Our findings demonstrate that STAT1 suppresses mammary tumor formation and its expression is frequently lost during breast cancer progression. Spontaneous mammary tumors that develop in STAT1-/- mice closely recapitulate the progression, ovarian hormone responsiveness, and molecular characteristics of human luminal breast cancer, the most common subtype of human breast neoplasms, and thus represent a valuable platform for testing novel treatments and detection modalities.