Generation of a Novel MMTV-tTA Transgenic Mouse Strain for the Targeted Expression of Genes in the Embryonic and Postnatal Mammary Gland

We have generated a new and improved transgenic mouse strain that permits a temporally controlled expression of transgenes throughout mammary gland development. High expression of the tetracycline-regulatible transactivator (tTA) under control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) is restricted to mammary epithelial cells and the salivary gland. The novel MMTV-tTA mouse strain induces a sustained transactivation of responder transgenes, which can be swiftly suppressed through administration of doxycycline (Dox). An important characteristic of this strain is its expression in early progenitor cells of mammary gland anlagen beginning at day 13.5 of embryonic development. We show here that the MMTV-tTA can be used in combination with GFP reporter strains to visualize CK8/CK14-dual positive progenitors in newborn females and their derived basal and luminal epithelial cell lineages in adult females. Our observations suggest that the novel MMTV-tTA can be utilized to express exogenous proteins in multipotent mammary progenitors during the earliest stages of mammary gland development to assess their biological significance throughout mammogenesis. Moreover, we demonstrate that the expression of the MMTV-tTA is sustained during mammary gland tumorigenesis in female mice expressing wildtype ErbB2. This makes this strain particular valuable to target the expression of exogenous proteins into developing mammary tumors to assess their significance in biological processes, such as tumor cell growth and survival, metabolism, and metastasis.     

Notch signaling regulates mammary stem cell function and luminal cell-fate commitment

The recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.     

RANK overexpression in transgenic mice with mouse mammary tumor virus promoter-controlled RANK increases proliferation and impairs alveolar differentiation in the mammary epithelia and disrupts lumen formation in cultured epithelial acini

RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins beta-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis.     

Mammary tumorigenesis in feral Mus cervicolor popaeus.

A pedigreed breeding population of feral Mus cervicolor popaeus with a high incidence of mammary tumors, arising between 6 and 14 months of age, is described. These mice were chronically infected with a type B retrovirus which is distantly related to the mouse mammary tumor virus (MMTV) of inbred strains of Mus musculus. MMTV-induced mammary tumors in inbred mice frequently (80%) contained an insertion of the viral genome into the int-1 or int-2 loci of the tumor cellular genome. These two cellular genetic loci were also altered by viral insertion in 11 of 20 M. cervicolor popaeus mammary tumor cellular DNAs tested. Results of our study of mammary tumorigenesis in feral mice demonstrate that viral-induced rearrangement and activation of the int loci are not limited to the genetic background of inbred mice selected for highly infectious MMTV and a high incidence of mammary tumors.     

Krt6a-Positive Mammary Epithelial Progenitors Are Not at Increased Vulnerability to Tumorigenesis Initiated by ErbB2

While most breast cancers are thought to arise from the luminal layer of the breast tissue, it remains unclear which specific cells in the luminal layer are the cells of origin of breast cancer. We have previously reported that WAP-positive luminal epithelial cells are at increased susceptibility to tumor initiation by ErbB2 compared to the bulk population, while the mammary cells with canonical Wnt signaling activity fail to evolve into tumors upon ErbB2 activation. Here, we used retrovirus to introduce ErbB2 into the Krt6a-positive mammary progenitor subset of the luminal epithelium and, for comparison, into the mammary luminal epithelium indiscriminately. Tumors developed from both groups of cells with a similar latency. These data indicate that the Krt6a-positive subset of mammary epithelial cells can be induced to form cancer by ErbB2 but it is not more susceptible to tumorigenesis initiated by ErbB2 than the bulk population of the luminal epithelium.     

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2^SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.     

Small Molecule Antagonists of the Wnt/Beta-Catenin Signaling Pathway Target Breast Tumor-Initiating Cells in a Her2/Neu Mouse Model of Breast Cancer

Background

Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure.

Methods/Findings

To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118–310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118–310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118–310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118–310 treated mice were unable to seed the growth of secondary tumors after transplant.

Conclusions

These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.     

Nonuniform expression of a mouse mammary tumor virus-driven int-2/Fgf-3 transgene in pregnancy-responsive breast tumors.

We have developed transgenic mice in which expression of the mouse int-2/Fgf-3 gene is regulated by a single long terminal repeat from mouse mammary tumor virus. Such mice contain and transmit a replica of the activated int-2/Fgf-3 allele present in a spontaneous mammary tumor from a BR6 mouse. Although free of infectious mouse mammary tumor virus and with a different genetic background, the transgenic mice develop pregnancy-responsive mammary epithelial proliferations that are similar to the early stages of tumorigenesis in the BR6 strain. Histological examination revealed that most of these tumors showed pronounced tubular and acinar structures, features usually associated with morphological differentiation. In some cases, the tumors were locally invasive, causing disruption of the dermis which manifested itself as local hair loss. In situ hybridization showed that patterns of transgene expression in the abnormal glands were markedly nonuniform. In contrast, mouse mammary tumor virus-induced neoplasms showed more uniform expression of int-2/Fgf-3, as did the urogenital epithelial proliferations that occur among males of this transgenic line. These data suggest that mammary tumors in virally infected animals may depend primarily on autocrine stimulation by the int-2/Fgf-3 gene product, whereas both autocrine and paracrine mechanisms may contribute to tumors and hyperplasias found in transgenic animals.     

Physiological levels of Pik3ca(H1047R) mutation in the mouse mammary gland results in ductal hyperplasia and formation of ERα-positive tumors

PIK3CA, the gene coding for the p110α subunit of phosphoinositide 3-kinase, is frequently mutated in a variety of human tumors including breast cancers. To better understand the role of mutant PIK3CA in the initiation and/or progression of breast cancer, we have generated mice with a conditional knock-in of the common activating mutation, Pik3ca(H1047R), into one allele of the endogenous gene in the mammary gland. These mice developed a ductal anaplasia and hyperplasia by 6 weeks of age characterized by multi-layering of the epithelial lining of the mammary ducts and expansion of the luminal progenitor (Lin(-); CD29(lo); CD24(+); CD61(+)) cell population. The Pik3ca(H1047R) expressing mice eventually develop mammary tumors with 100% penetrance but with a long latency (>12 months). This is significantly longer than has been reported for transgenic models where expression of the mutant Pik3ca is driven by an exogenous promoter. Histological analysis of the tumors formed revealed predominantly ERα-positive fibroadenomas, carcinosarcomas and sarcomas. In vitro induction of Pik3ca(H1047R) in immortalized mammary epithelial cells also resulted in tumor formation when injected into the mammary fat pad of immunodeficient recipient mice. This novel model, which reproduces the scenario of a heterozygous somatic mutation occurring in the endogenous PIK3CA gene, will thus be a valuable tool for investigating the role of Pik3ca(H1047R) mutation in mammary tumorigenesis both in vivo and in vitro.     

Genetic Mechanisms in Apc-Mediated Mammary Tumorigenesis

Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; Apc^CKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC.