The basal-like mammary carcinomas induced by Brca1 or Bard1 inactivation implicate the BRCA1/BARD1 heterodimer in tumor suppression

Women with germ-line mutations of the BRCA1 tumor suppressor gene are highly susceptible to breast and ovarian cancer. The protein product of BRCA1 is involved in a broad spectrum of biological processes and interacts with many diverse proteins. One of these, BARD1, associates with BRCA1 to form a heterodimeric complex that is enzymatically active as an ubiquitin E3 ligase. Although the BRCA1/BARD1 heterodimer has been implicated in several aspects of BRCA1 function, its role in tumor suppression has not been evaluated. To address this question, we generated mouse strains carrying conditional alleles of either Bard1 or Brca1 and used Cre recombination to inactivate these genes in mammary epithelial cells. Significantly, the conditional Bard1- and Brca1-mutant mice developed breast carcinomas that are indistinguishable from each other (and from those of double conditional Bard1/Brca1-mutant animals) with respect to their frequency, latency, histopathology, and cytogenetic features. Reminiscent of the basal-like breast carcinomas seen in human BRCA1 mutation carriers, these tumors are "triple negative" for estrogen and progesterone receptor expression and HER2/neu amplification. They also express basal cytokeratins CK5 and CK14, have an elevated frequency of p53 lesions, and display high levels of chromosomal instability. The remarkable similarities between the mammary carcinomas of Bard1-, Brca1-, and Bard1/Brca1-mutant mice indicate that the tumor suppressor activities of both genes are mediated through the BRCA1/BARD1 heterodimer.     

Expression of Q227L-Gαs in MCF-7 human breast cancer cells inhibits tumorigenesis

The effects of expression of mutant (Q227L)-activated Gα_s and elevation of cAMP on mitogen-activating protein kinase (MAPK) activity and the transformed phenotype were studied in the MCF-7 human mammary epithelial cell line. Elevation of cAMP partially inhibited the epidermal growth factor-stimulated DNA synthesis and the intrinsic MAPK (ERK-1 and ERK-2) of serum-starved MCF-7 cells. Addition of 8Br-cAMP or expression of mutant (Q227L)-activated Gα_s in MCF-7 cells blocked the ability of these cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. 8Br-cAMP in the culture medium also blocked estrogen stimulation of MCF-7 cell proliferation in vitro. MCF-7 cells expressing Q227L-Gα_s grew very slowly in vitro, and when these cells were injected s.c. into athymic mice implanted with estrogen pellets, the frequency of tumor formation was reduced greatly and the sizes of the tumors formed were much smaller than those in mice injected with MCF-7 cells that had been transfected with the empty vector. These results indicate that the intracellular levels of cAMP in transformed mammary epithelial cells can be a crucial factor in determining the expression of the transformed phenotype. Interactions between the G_s/adenylyl cyclase and MAPK-1,2 signaling pathways could be one mechanism by which expression of the transformed phenotype in mammary epithelial cells are regulated.     

Palb2 synergizes with Trp53 to suppress mammary tumor formation in a model of inherited breast cancer

Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or to Fanconi Anemia subtype N. PALB2 performs its tumor suppressor role, at least in part, by supporting homologous recombination-type double strand break repair (HR-DSBR) through physical interactions with BRCA1, BRCA2, and RAD51. To further understand the mechanisms underlying PALB2-mediated DNA repair and tumor suppression functions, we targeted Palb2 in the mouse. Palb2-deficient murine ES cells recapitulated DNA damage defects caused by PALB2 depletion in human cells, and germ-line deletion of Palb2 led to early embryonic lethality. Somatic deletion of Palb2 driven by K14-Cre led to mammary tumor formation with long latency. Codeletion of both Palb2 and Tumor protein 53 (Trp53) accelerated mammary tumor formation. Like BRCA1 and BRCA2 mutant breast cancers, these tumors were defective in RAD51 focus formation, reflecting a defect in Palb2 HR-DSBR function, a strongly suspected contributor to Brca1, Brca2, and Palb2 mammary tumor development. However, unlike the case of Brca1-mutant cells, Trp53bp1 deletion failed to rescue the genomic instability of Palb2- or Brca2-mutant primary lymphocytes. Therefore, Palb2-driven DNA damage control is, in part, distinct from that executed by Brca1 and more similar to that of Brca2. The mechanisms underlying Palb2 mammary tumor suppression functions can now be explored genetically in vivo.     

Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer

Women carrying germ-line mutations in BRCA1 are strongly predisposed to developing breast cancers with characteristic features also observed in sporadic basal-like breast cancers. They appear as high-grade tumors with high proliferation rates and pushing borders. On the molecular level, they are negative for hormone receptors and ERBB2, display frequent TP53 mutations, and express basal epithelial markers. To study the role of BRCA1 and P53 loss of function in breast cancer development, we generated conditional mouse models with tissue-specific mutation of Brca1 and/or p53 in basal epithelial cells. Somatic loss of both BRCA1 and p53 resulted in the rapid and efficient formation of highly proliferative, poorly differentiated, estrogen receptor-negative mammary carcinomas with pushing borders and increased expression of basal epithelial markers, reminiscent of human basal-like breast cancer. BRCA1- and p53-deficient mouse mammary tumors exhibit dramatic genomic instability, and their molecular signatures resemble those of human BRCA1-mutated breast cancers. Thus, these tumors display important hallmarks of hereditary breast cancers in BRCA1-mutation carriers.     

Cell–cell interaction in prostate gene regulation and cytodifferentiation

To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by flow cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57. Basal epithelial cells express CD44, and luminal epithelial cells express CD57. The CD57⁺ luminal cells are the terminally differentiated secretory cells of the gland that synthesize prostate-specific antigen (PSA). Expression of PSA is regulated by androgen, and PSA mRNA is one of the abundant messages in these cells. We show that PSA expression by the CD57⁺ cells is abolished after prostate tissue is dispersed by collagenase into single cells. Expression is restored when CD57⁺ cells are reconstituted with stromal cells. The CD44⁺ basal cells possess characteristics of stem cells and are the candidate progenitors of luminal cells. Differentiation, as reflected by PSA production, can be detected when CD44⁺ cells are cocultured with stromal cells. Our studies show that cell–cell interaction plays an important role in prostatic cytodifferentiation and the maintenance of the differentiated state.     

Inducible expression of dominant negative insulin-like growth factor I receptor in MCF-7 breast cancer cells

The insulin-like growth factor I receptor (IGF-IR) is expressed in many cell types and is critical for normal growth and development. In the healthy mammary gland, the role of IGF-IR is not fully elucidated. However, IGF-IR, which is primarily expressed in the mammary epithelial cells, is known to play an obligatory role in cellular transformation, facilitating the progression to breast cancer. We have utilized the tetracycline regulatory (tet-on) system to generate an in vitro model system to allow us to further investigate IGF-I/IGF-IR function in mammary epithelial cells. A plasmid construct containing a mutant IGF-I receptor (IGF-IR-DN) fused to the tetracycline operator (tetOPh_CMV-IGF-IR-DN) was stably transfected into MCF-7 human breast cancer cells. The conditional regulation of the IGF-IR-DN gene expression was studied in four independent clonal lines. The translated IGF-IR-DN protein was detected only in the stably transfected doxycycline-induced cells, and its expression was up-regulated (three- to sixfold) following induction. IGF-I stimulated cell proliferation diminished (twofold) in doxycycline-induced cells compared to uninduced cells, demonstrating that the transgene construct was functional and ruling out any pleiotropic effect that may be attributed to doxycycline. Interestingly, autophosphorylation of the IGF-IR and phosphorylation of the downstream substrate, insulin receptor substrate-1 (IRS-1), was not inhibited in doxycycline/IGF-I treated cells, suggesting the possibility that activation of downstream substrates other than the IRS-1 may be critical for optimal cell proliferation. This novel in vitro model should allow us to more directly examine the role of IGF-I/IGF-IR signaling and function in mammary epithelial cells.     

Posttranslational regulation of cyclin D1 by retinoic acid: A chemoprevention mechanism

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G₁ in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G₁-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G₁ active in the prevention of human bronchial epithelial cell transformation.     

Inhibition of granulocytic differentiation by mNotch1

Effective hematopoiesis requires the commitment of pluripotent and multipotent stem cells to distinct differentiation pathways, proliferation and maturation of cells in the various lineages, and preservation of pluripotent progenitors to provide continuous renewal of mature blood cells. While the importance of positive and negative cytokines in regulating proliferation and maturation of hematopoietic cells has been well documented, the factors and molecular processes involved in lineage commitment and self-renewal of multipotent progenitors have not yet been defined. In other developmental systems, cellular interactions mediated by members of the Notch gene family have been shown to influence cell fate determination by multipotent progenitors. We previously described the expression of the human Notch1 homolog, TAN-1, in immature hematopoietic precursors. We now demonstrate that constitutive expression of the activated intracellular domain of mouse Notch1 in 32D myeloid progenitors inhibits granulocytic differentiation and permits expansion of undifferentiated cells, findings consistent with the known function of Notch in other systems.     

Increased sensitivity to apoptotic stimuli in c-abl-deficient progenitor B-cell lines

The protooncogene c-abl encodes a nonreceptor tyrosine kinase whose cellular function is unknown. To study the possible involvement of c-Abl in proliferation, differentiation, and cell cycle regulation of early B cells, long-term lymphoid bone marrow cultures were established from c-abl-deficient mice and their wild-type littermates. Interleukin 7-dependent progenitor B-cell clones and lines expressing B220 and CD43 could be generated from both mutant and wild-type mice. The mutant and wild-type lines displayed no difference in their proliferative capacity as measured by thymidine incorporation in response to various concentrations of interleukin 7. Similarly, c-abl deficiency did not interfere with the ability of mutant clones to differentiate into surface IgM-positive cells in vitro. Analysis of cultures after growth factor deprivation, however, revealed a strikingly accelerated rate of cell death in c-abl mutant cells, due to apoptosis as confirmed by terminal deoxynucleotidyltransferase-mediated UTP nick end labeling analysis. Furthermore, a greater susceptibility to apoptotic cell death in c-abl mutant cells was also observed after glucocorticoid treatment. These results suggest that mutant c-Abl renders the B-cell progenitors more sensitive to apoptosis, and may account for some of the phenotypes observed in c-abl-deficient animals.     

Increased LHX9 expression in alveolar epithelial type 2 cells of patients with chronic obstructive pulmonary disease

BackgroundAlveolar epithelial type 2 (AT2) cells serve as stem cells in alveolar epithelium and are assumed to lose their stem cell function in the lungs of chronic obstructive pulmonary disease (COPD). Although we previously reported that LHX9 mRNA expression was up-regulated in AT2 cells of COPD lung tissues, it is yet to be elucidated how LHX9 is associated with the vulnerability of AT2 cells in COPD.MethodsAT2 cells were isolated from lung tissues of 10 non-COPD subjects and 11 COPD patients. LHX9 mRNA expression was determined by quantitative RT-PCR. To identify up-stream molecules, an alveolar epithelial cell line A549 was exposed to pro-inflammatory cytokines in vitro. siRNA-mediated Lhx9 knockdown was performed to determine how Lhx9 affected the cellular viability and the cell-division cycle.ResultsLHX9 mRNA expression was increased in AT2 cells from COPD lung tissues, compared to those from non-COPD tissues. The airflow obstruction was independently correlated with the increase in LHX9 expression. Among several pro-inflammatory cytokines, interferon-γ was a strong inducer of LHX9 expression in A549 cells. Lhx9 was involved in the increased susceptibility to serum starvation-induced death of A549 cells.ConclusionsOur data suggest that IFN-γ predominantly increases the LHX9 expression which enhances the susceptibility to cell death. Considering the independent association of the increased LHX9 expression in AT2 cells with airflow obstruction, the IFN-γ-Lhx9 axis might contribute to the vulnerability of AT2 cells in the lungs of COPD patients.     

Expression of the insulin-like growth factor binding proteins during postnatal development of the murine mammary gland

IGF-I and IGF-II have known roles in postnatal development of the mammary gland. In contrast, the function of the high-affinity IGF binding proteins (IGFBPs) in mammary growth and differentiation is largely unknown. The goal of these studies was to determine the patterns and levels of IGFBP expression during postnatal growth of the murine mammary gland. IGFBP-1 to -5 proteins were detected in mammary tissue by immunoblotting during both pubertal and pregnancy-induced growth; however, the regulation of each IGFBP was distinct through these developmental periods. IGFBP-2 to -5 mRNAs were readily detectable in the developing gland by in situ hybridization analyses but were expressed in distinct cellular sites. IGFBP-3 and -5 mRNAs were expressed in the developing epithelial structures and in isolated stromal cells during ductal growth and alveolar differentiation. In the terminal end buds (TEBs), IGFBP-3 mRNA expression was consistent with its localization in the cap cells, whereas IGFBP-5 was highly expressed in the body cells of the TEB. In contrast, IGFBP-2 and -4 mRNAs were expressed predominantly in stromal cells. IGFBP-2 mRNA was localized to restricted sites in the neck of the TEB and along the ductal structures, whereas IGFBP-4 mRNA was widely expressed in the stroma surrounding the epithelial structures. Protein and mRNA expression for most of the IGFBPs decreased during lactational ages. Levels of IGFBP-2 and -5 protein increased after pup removal during forced involution. Taken together, these data suggest important functions for the family of IGFBPs during postnatal growth and differentiation of the mammary epithelium.     

Resistance to apoptosis in CTLL-2 cells constitutively expressing c-Myb is associated with induction of BCL-2 expression and Myb-dependent regulation of bcl-2 promoter activity

c-Myb, the cellular homologue of the transforming gene of the avian myeloblastosis virus, is preferentially expressed in all hematopoietic lineages, including T and B lymphocyte lineages. In T lymphocytes, c-Myb expression appears to be required for cell cycle progression and proliferation. To further investigate the role of c-Myb in T cell proliferation and survival, interleukin (IL) 2-dependent CTLL-2 cells were transfected with a constitutively active c-myb or with a c-myb antisense construct able to down-regulate endogenous Myb levels, and the transfectants were assessed for proliferation and survival in low concentrations of IL-2 and for susceptibility to dexamethasone-induced apoptosis. Compared with control cells, CTLL-2 cells constitutively expressing c-Myb proliferate in low concentrations of IL-2 and are less susceptible to apoptosis induced by IL-2 deprivation or treatment with dexamethasone. In contrast, cells transfected with an antisense c-myb construct do not proliferate in low concentrations of IL-2 and undergo apoptosis upon IL-2 deprivation or dexamethasone treatment more rapidly than parental cells. Overexpression of c-Myb was accompanied by up-regulation of BCL-2 expression. In transient transfection assays, the murine bcl-2 promoter was efficiently transactivated by c-Myb, but such effect was observed also in cells transfected with a DNA binding-deficient c-myb construct. Moreover, in gel retardation assays, a 38-bp oligomer in the shortest bcl-2 promoter segment regulated by c-Myb formed a specific complex with nuclear extracts from c-Myb-transfected CTLL-2 cells. Thus, these results strongly suggest that c-Myb, in addition to regulating T cell proliferation, protects T lymphocytes from apoptosis by induction of BCL-2 expression, which involves a c-Myb-dependent mechanism of promoter regulation.     

Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasite Theileria parva

When T cells become infected by the parasite Theileria parva, they acquire a transformed phenotype and no longer require antigen-specific stimulation or exogenous growth factors. This is accompanied by constitutive interleukin 2 (IL-2) and IL-2 receptor expression. Transformation can be reversed entirely by elimination of the parasites using the specific drug BW720c. Extracellular signal-regulated kinase and jun NH2-terminal kinase (JNK) are members of the mitogen-activated protein kinase family, which play a central role in the regulation of cellular differentiation and proliferation and also participate in the regulation of IL-2 and IL-2 receptor gene expression. T. parva was found to induce an unorthodox pattern of mitogen-activated protein kinase expression in infected T cells. JNK-1 and JNK-2 are constitutively active in a parasite-dependent manner, but have altered properties. In contrast, extracellular signal-regulated kinase-2 is not activated even though its activation pathway is functionally intact. Different components of the T cell receptor (TCR)-dependent signal transduction pathways also were examined. The TCRζ or CD3 chains were found not to be phosphorylated and T. parva-transformed T cells were resistant to inhibitors that block the early steps of T cell activation. Compounds that inhibit the progression of T cells to proliferation, however, were inhibitory. Our data provide the first example, to our knowledge, for parasite-mediated JNK activation, and our findings strongly suggest that T. parva not only lifts the requirement for antigenic stimulation but also entirely bypasses early TCR-dependent signal transduction pathways to induce continuous proliferation.     

Autocrine growth hormone prevents lactogenic differentiation of mouse mammary epithelial cells

We have examined the expression, postnatal ontogeny, and localization of mouse GH (mGH) and its relative expression during pregnancy, lactation, and weaning in the mouse. mGH mRNA and protein was expressed predominantly in the epithelial component of the mammary gland, and maximal expression was observed during the pubertal period. Autocrine mGH expression dramatically decreased during late pregnancy and lactation. Concordantly, autocrine mGH expression is repressed during forced differentiation of mouse HC11 mammary epithelial cells in culture. Forced expression of mGH in HC11 cells abrogated lactogenic differentiation as indicated by reduced expression of beta-casein and reduced expression and loss of lateral epithelial localization of E-cadherin. Forced expression of mGH in mouse mammary epithelial cells increased cell survival and proliferation and consequently increased the size of mammary acinar-like structures formed in three-dimensional Matrigel. Thus, autocrine mGH expression in the mouse mammary epithelial cell is maximal at puberty and prevents mammary epithelial cell differentiation. Autocrine GH will therefore participate in mammary morphogenic processes at puberty.