The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment.

Amplification and overexpression of erbB2/neu proto-oncogene is observed in 20-30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2 in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2 because of constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2ΔEx16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16-derived mammary tumors exhibit several unique features that distinguish this model from the conventional ErbB2 ones expressing the erbB2 proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2-derived tumors that express luminal keratins, ErbB2ΔEx16-derived tumors exhibit high degree of intratumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibit distinct signaling and gene expression profiles that correlate with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal.

Lactate detection in inducible and orthotopic Her2/neu mammary gland tumours in mouse models.

This study compared the steady state concentration of lactate in an inducible Her2/nue transgenic breast cancer mouse model and in tumours from the same Her2/neu model grown orthotopically. In vivo lactate was detected by MRS using the Hadamard encoded selective multiple quantum coherence pulse sequence (HadSelMQC) recently developed by our laboratory. A lower lactate signal was observed in the inducible tumours compared to orthotopic tumours in vivo, while ex vivo analysis of perchloric acid extracts revealed similar amounts of this metabolite in both models. Histological staining of mammary tumour specimens showed a much higher level of fat tissue in inducible tumours compared to the orthotopic model. Phantom studies with [3-(13) C] lactate indicated that a lipid environment could significantly reduce the T2 of lactate and impede its detection. The transgenic inducible model for breast cancer not only better recapitulated the biological aspects of the human disease but also provided additional characteristics related to in vivo detection of lactate that are not available in orthotopic or xenograft models. This study suggests that the level of lactate measured by the HadSelMQC pulse sequence may be underestimated in human patients in the presence of high lipid levels that are typically encountered in the breast.

Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden.

The transcription factor nuclear factor kappa B (NF-κB) is activated in human breast cancer tissues and cell lines. However, it is unclear whether NF-κB activation is a consequence of tumor formation or a contributor to tumor development. We developed a doxycycline (dox)-inducible mouse model, termed DNMP, to inhibit NF-κB activity specifically within the mammary epithelium during tumor development in the polyoma middle T oncogene (PyVT) mouse mammary tumor model. DNMP females and PyVT littermate controls were treated with dox from 4 to 12 weeks of age. We observed an increase in tumor latency and a decrease in final tumor burden in DNMP mice compared with PyVT controls. A similar effect with treatment from 8 to 12 weeks indicates that outcome is independent of effects on postnatal virgin ductal development. In both cases, DNMP mice were less likely to develop lung metastases than controls. Treatment from 8 to 9 weeks was sufficient to impact primary tumor formation. Inhibition of NF-κB increases apoptosis in hyperplastic stages of tumor development and decreases proliferation at least in part by reducing Cyclin D1 expression. To test the therapeutic potential of NF-κB inhibition, we generated palpable tumors by orthotopic injection of PyVT cells and then treated systemically with the NF-κB inhibitor thymoquinone (TQ). TQ treatment resulted in a reduction in tumor volume and weight as compared with vehicle-treated control. These data indicate that epithelial NF-κB is an active contributor to tumor progression and demonstrate that inhibition of NF-κB could have a significant therapeutic impact even at later stages of mammary tumor progression.

The Ras oncogene signals centrosome amplification in mammary epithelial cells through cyclin D1/Cdk4 and Nek2.

Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-Ras(G12D) and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-Ras(G12D), it was detected only in premalignant mammary lesions expressing K-Ras(G12D). CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-Ras(G12V) abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA.

Transgenic overexpression of IGF-IR disrupts mammary ductal morphogenesis and induces tumor formation.

Overexpression and hyperactivation of the type I insulin-like growth factor receptor (IGF-IR) has been observed in human breast tumor biopsies. In addition, in vitro studies indicate that overexpression of IGF-IR is sufficient to transform cells such as mouse embryo fibroblasts and this receptor promotes proliferation and survival in breast cancer cell lines. To fully understand the function of the IGF-IR in tumor initiation and progression, transgenic mice containing human IGF-IR under a doxycycline-inducible MMTV promoter system were generated. Administration of 2 mg/ml doxycycline in the animals' water supply beginning at 21 days of age resulted in elevated levels of IGF-IR in mammary epithelial cells as detected by Western blotting and immunohistochemistry. Whole mount analysis of 55-day-old mouse mammary glands revealed that IGF-IR overexpression significantly impaired ductal elongation. Moreover, histological analyses revealed multiple hyperplasic lesions in the mammary glands of these 55-day-old mice. The formation of palpable mammary tumors was evident at approximately 2 months of age and was associated with increased levels of IGF-IR signaling molecules including phosphorylated Akt, Erk1/Erk2 and STAT3. Therefore, these transgenic mice provide evidence that IGF-IR overexpression is sufficient to induce mammary epithelial hyperplasia and tumor formation in vivo and provide a model to further understand the function of IGF-IR in mammary epithelial transformation.

Characterization of adjacent breast tumors using oligonucleotide microarrays.

BACKGROUND: Current methodology often cannot distinguish second primary breast cancers from multifocal disease, a potentially important distinction for clinical management. In the present study we evaluated the use of oligonucleotide-based microarray analysis in determining the clonality of tumors by comparing gene expression profiles. METHOD: Total RNA was extracted from two tumors with no apparent physical connection that were located in the right breast of an 87-year-old woman diagnosed with invasive ductal carcinoma (IDC). The RNA was hybridized to the Affymetrix Human Genome U95A Gene Chip (12,500 known human genes) and analyzed using the Gene Chip Analysis Suite 3.3 (Affymetrix, Inc, Santa Clara, CA, USA) and JMPIN 3.2.6 (SAS Institute, Inc, Cary, NC, USA). Gene expression profiles of tumors from five additional patients were compared in order to evaluate the heterogeneity in gene expression between tumors with similar clinical characteristics. RESULTS: The adjacent breast tumors had a pairwise correlation coefficient of 0.987, and were essentially indistinguishable by microarray analysis. Analysis of gene expression profiles from different individuals, however, generated a pairwise correlation coefficient of 0.710. CONCLUSION: Transcriptional profiling may be a useful diagnostic tool for determining tumor clonality and heterogeneity, and may ultimately impact on therapeutic decision making.

Analysis of murine Brca2 reveals conservation of protein-protein interactions but differences in nuclear localization signals.

In this report, we have analyzed the protein encoded by the murine Brca2 locus. We find that murine Brca2 shares multiple properties with human BRCA2 including its regulation during the cell cycle, localization to nuclear foci, and interaction with Brca1 and Rad51. Murine Brca2 stably interacts with human BRCA1, and the amino terminus of Brca2 is sufficient for this interaction. Exon 11 of murine Brca2 is required for its stable association with RAD51, whereas the carboxyl terminus of Brca2 is dispensable for this interaction. Finally, in contrast to human BRCA2, we demonstrate that carboxyl-terminal truncations of murine Brca2 localize to the nucleus. This finding may explain the apparent inconsistency between the cytoplasmic localization of carboxyl-terminal truncations of human BRCA2 and the hypomorphic phenotype of mice homozygous for similar carboxyl-terminal truncating mutations.

Impaired DNA damage response in cells expressing an exon 11-deleted murine Brca1 variant that localizes to nuclear foci.

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-Delta11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-Delta11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein. Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-Delta11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-Delta11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-Delta11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-Delta11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-Delta11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.

c-MYC induces mammary tumorigenesis by means of a preferred pathway involving spontaneous Kras2 mutations.

Although the process of mammary tumorigenesis requires multiple genetic events, it is unclear to what extent carcinogenesis proceeds through preferred secondary pathways following a specific initiating oncogenic event. Similarly, the extent to which established mammary tumors remain dependent on individual mutations for maintenance of the transformed state is unknown. Here we use the tetracycline regulatory system to conditionally express the human c-MYC oncogene in the mammary epithelium of transgenic mice. MYC encodes a transcription factor implicated in multiple human cancers. In particular, amplification and overexpression of c-MYC in human breast cancers is associated with poor prognosis, although the genetic mechanisms by which c-MYC promotes tumor progression are poorly understood. We show that deregulated c-MYC expression in this inducible system results in the formation of invasive mammary adenocarcinomas, many of which fully regress following c-MYC deinduction. Approximately half of these tumors harbor spontaneous activating point mutations in the ras family of proto-oncogenes with a strong preference for Kras2 compared with Hras1. Nearly all tumors lacking activating ras mutations fully regressed following c-MYC deinduction, whereas tumors bearing ras mutations did not, suggesting that secondary mutations in ras contribute to tumor progression. These findings demonstrate that c-MYC-induced mammary tumorigenesis proceeds through a preferred secondary oncogenic pathway involving Kras2.

The caM kinase, Pnck, is spatially and temporally regulated during murine mammary gland development and may identify an epithelial cell subtype involved in breast cancer.

While screening for protein kinases expressed in the murine mammary gland, we identified previously a Ca2+/calmodulin-dependent kinase, Pnck, that is most closely related to CaMKI. In this report, we show that Pnck is temporally regulated during murine mammary development with highest levels of expression observed late in pregnancy, concomitant with the decreased cellular proliferation and terminal differentiation of the mammary epithelium. Consistent with this finding, Pnck is up-regulated in confluent mammary epithelial cells and is down-regulated as serum-starved cells are stimulated to reenter the cell cycle. In the mammary gland, Pnck is expressed in an epithelial-specific and markedly heterogeneous manner, suggesting that the expression of this kinase may be restricted to a particular mammary epithelial cell type. Potentially related to its heterogeneous in vivo expression pattern, Pnck expression is oncogene-associated in murine epithelial cell lines derived from mammary tumors arising in different transgenic mouse models of breast cancer; cell lines derived from mammary tumors initiated by c-myc or int-2/Fgf3 express Pnck, whereas cell lines initiated by neu or H-ras do not. In an analogous manner, expression of the human homologue of Pnck is restricted to a subset of human breast cancer cell lines. Moreover, PNCK was found to be highly overexpressed in a subset of human primary human breast cancers compared with benign mammary tissue. Together, our data suggest that Pnck may play a role in mammary development, and that expression of this kinase may be restricted to a mammary epithelial cell type that is transformed in a subset of human breast cancers.

Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland.

The steroid hormones 17 beta-estradiol and progesterone play a central role in the pathogenesis of breast cancer and regulate key phases of mammary gland development. This suggests that developmental regulatory molecules whose activity is influenced by ovarian hormones may also contribute to mammary carcinogenesis. In a screen designed to identify protein kinases expressed in the mammary gland, we previously identified a novel SNF1-related serine/threonine kinase, Hunk (hormonally upregulated Neu-associated kinase). During postnatal mammary development, Hunk mRNA expression is restricted to a subset of mammary epithelial cells and is temporally regulated with highest levels of expression occurring during early pregnancy. In addition, treatment of mice with 17 beta-estradiol and progesterone results in the rapid and synergistic upregulation of Hunk expression in a subset of mammary epithelial cells, suggesting that the expression of this kinase may be regulated by ovarian hormones. Consistent with the tightly regulated pattern of Hunk expression during pregnancy, mammary glands from transgenic mice engineered to misexpress Hunk in the mammary epithelium manifest temporally distinct defects in epithelial proliferation and differentiation during pregnancy, and fail to undergo normal lobuloalveolar development. Together, these observations suggest that Hunk may contribute to changes in the mammary gland that occur during pregnancy in response to ovarian hormones.

Protein kinase expression during murine mammary development.

The susceptibility of the mammary gland to carcinogenesis is influenced by its normal development, particularly during developmental stages such as puberty and pregnancy that are characterized by marked changes in proliferation and differentiation. Protein kinases are important regulators of proliferation and differentiation, as well as of neoplastic transformation, in a wide array of tissues, including the breast. Using a RT-PCR-based cloning strategy, we have identified 41 protein kinases that are expressed in breast cancer cell lines and in the murine mammary gland during development. The expression of each of these kinases was analyzed throughout postnatal mammary gland development as well as in a panel of mammary epithelial cell lines derived from distinct transgenic models of breast cancer. Although the majority of protein kinases isolated in this screen have no currently recognized role in mammary development, most kinases examined were found to exhibit developmental regulation. After kinases were clustered on the basis of similarities in their temporal expression profiles during mammary development, multiple distinct patterns of expression were observed. Analysis of these patterns revealed an ordered set of expression profiles in which successive waves of kinase expression occur during development. Interestingly, several protein kinases whose expression has previously been reported to be restricted to tissues other than the mammary gland were isolated in this screen and found to be expressed in the mammary gland. In aggregate, these findings suggest that the array of kinases participating in the regulation of normal mammary development is considerably broader than currently appreciated.

Cloning and characterization of Hunk, a novel mammalian SNF1-related protein kinase.

We previously identified a novel protein kinase, Hunk, by means of a degenerate PCR screen designed to isolate kinases expressed in the murine mammary gland. We now describe the molecular cloning, chromosomal localization, and activity of this kinase and characterize its spatial and temporal pattern of expression in the mouse. We have isolated a 5.0-kb full-length cDNA clone that contains the 714-amino-acid open reading frame encoding Hunk. Analysis of this cDNA reveals that Hunk is most closely related to the SNF1 family of serine/threonine kinases and contains a newly described SNF1 homology domain. Accordingly, antisera specific for Hunk detect an 80-kDa polypeptide with associated phosphotransferase activity. Hunk is located on distal mouse chromosome 16 in a region of conserved synteny with human chromosome 21q22. During fetal development and in the adult mouse, Hunk mRNA expression is developmentally regulated and tissue-specific. Moreover, in situ hybridization analysis reveals that Hunk expression is restricted to subsets of cells within a variety of organs in the adult mouse. These findings suggest a role for Hunk in murine development.

Mammary gland development, reproductive history, and breast cancer risk.

The observation that normal pathways of differentiation and development are invariably altered during the process of carcinogenesis implies an intrinsic relationship between these processes. This relationship is particularly evident in the breast, as exemplified by the existence of endocrine risk factors for breast cancer that are related to the timing of normal developmental events. Understanding the mechanisms by which normal developmental events alter breast cancer risk is a central focus of our laboratory. Herein, we describe three approaches being taken in our laboratory toward defining the molecular basis of this relationship. These include: determining the roles played by the tumor suppressor genes, BRCA1 and BRCA2, in the normal differentiation and development of the breast; studying the function of three novel protein kinases identified in our laboratory in mammary epithelial development; and defining the molecular and cellular changes that occur in the breast as a result of reproductive events known to influence breast cancer risk.

Cloning and characterization of Krct, a member of a novel subfamily of serine/threonine kinases.

Protein kinases frequently play key roles in the normal regulation of growth and development in eukaryotic organisms. As a consequence, aberrant expression or mutations in this family of molecules frequently result in transformation. Previously, we have conducted a screen to identify protein kinases that are expressed in the mouse during mammary gland development and in breast cancer cell lines. We now describe the molecular cloning, characterization and expression of Krct, a novel serine/threonine protein kinase unrelated to previously defined families of protein kinases. At the mRNA level, Krct is widely expressed throughout murine development and in adult tissues. Despite its ubiquitous expression, Krct is expressed preferentially within specific cellular compartments in multiple tissues, in particular within the testis and gastrointestinal tract. At the amino acid level, Krct is most closely related to four previously undescribed kinases in Saccharomyces cerevisiae, Arabidopsis thaliana and Caenorhabditis elegans. Together, these kinases appear to define a novel subfamily of serine/threonine protein kinases. Krct possesses an unusually long 5'-untranslated region containing multiple upstream initiation codons and, in this regard, is similar to many proto-oncogenes that regulate normal growth and differentiation. In addition, Krct is located on mouse chromosome 11 closely linked to the epidermal growth factor receptor and, therefore, is likely to be co-amplified in a variety of human tumors.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

Expression of BRCA1 and BRCA2 in normal and neoplastic cells.

Current evidence strongly supports a role for the breast cancer susceptibility genes, BRCA1 and BRCA2, in both normal development and carcinogenesis. Valuable clues regarding the function of these genes have been garnered through studies of their patterns of expression. A central feature of the in vivo pattern of BRCA1 and BRCA2 expression is that each of these putative tumor suppressor genes is expressed at maximal levels in rapidly proliferating cells. This feature is consistent with in vitro observations that BRCA1 and BRCA2 are expressed in a cell cycle-dependent manner. This feature is also well illustrated during mammary gland development wherein the expression of BRCA1 and BRCA2 is induced in rapidly proliferating cellular compartments undergoing differentiation, such as terminal end buds during puberty and developing alveoli during pregnancy. Strikingly, the spatial and temporal patterns of BRCA1 and BRCA2 expression are virtually indistinguishable during embryonic development and in multiple adult tissues despite the fact that these genes are unrelated. These observations have contributed to the emerging hypothesis that these genes function in similar regulatory pathways.

Precocious mammary gland development in P-cadherin-deficient mice.

To investigate the functions of P-cadherin in vivo, we have mutated the gene encoding this cell adhesion receptor in mice. In contrast to E- and N-cadherin- deficient mice, mice homozygous for the P-cadherin mutation are viable. Although P-cadherin is expressed at high levels in the placenta, P-cadherin-null females are fertile. P-cadherin expression is localized to the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. The role of the myoepithelium as a contractile tissue necessary for milk secretion is clear, but its function in the nonpregnant animal is unknown. The ability of the P-cadherin mutant female to nurse and maintain her litter indicates that the contractile function of the myoepithelium is not dependent on the cell adhesion molecule P-cadherin. The virgin P-cadherin-null females display precocious differentiation of the mammary gland. The alveolar-like buds in virgins resemble the glands of an early pregnant animal morphologically and biochemically (i.e., milk protein synthesis). The P-cadherin mutant mice develop hyperplasia and dysplasia of the mammary epithelium with age. In addition, abnormal lymphocyte infiltration was observed in the mammary glands of the mutant animals. These results indicate that P-cadherin-mediated adhesion and/or signals derived from cell-cell interactions are important determinants in negative growth control in the mammary gland. Furthermore, the loss of P-cadherin from the myoepithelium has uncovered a novel function for this tissue in maintaining the undifferentiated state of the underlying secretory epithelium.

Developmental expression of Brca2 colocalizes with Brca1 and is associated with proliferation and differentiation in multiple tissues.

Germline mutations in the putative tumor suppressor gene, BRCA1, predispose women to dramatically elevated risks of breast cancer, while germline mutations in the structurally unrelated gene, BRCA2, predispose both men and women to breast cancer. Recent studies have suggested an important developmental role for the murine homologue of BRCA1 in the regulation of proliferation and differentiation. At the present time, however, little is known about the developmental role of BRCA2 or the regulation of its expression in vivo. We have determined the spatial and temporal pattern of expression of the murine homologue of BRCA2 during fetal development, in adult tissues, and in the mammary gland during postnatal development. Our results indicate that Brca2 mRNA expression is highest in proliferating cellular compartments, particularly those undergoing differentiation. In the breast, Brca2 expression is developmentally regulated and is induced during puberty and pregnancy and as a result of parity. Surprisingly, in multiple fetal and adult tissues the spatial and temporal pattern of Brca2 mRNA expression is virtually indistinguishable from that of Brca1, despite the fact that these genes display no homology. These observations suggest that Brca2 is involved in the processes of proliferation and differentiation in the mammary gland and other tissues, and that Brca1 and Brca2 mRNA expression may be regulated by similar pathways and stimuli in multiple cell types. Interestingly, however, our analysis reveals that Brca1 and Brca2 expression are differentially regulated during the development of specific endocrine target tissues, such as the testis during spermatogenesis and the breast during pregnancy. In addition, the ratio of mRNA expression in the mammary glands of adult females relative to adult males is significantly greater for Brca1 than for Brca2. These observations imply that Brca1 and Brca2 mRNA expression are differentially regulated by sex hormones. In order to test this hypothesis, we have analyzed the expression of these two breast cancer susceptibility genes in ovariectomized mice treated with 17beta-estradiol and progesterone. Our results demonstrate that the up-regulation of mRNA expression in the breast by ovarian hormones is significantly greater for Brca1 than for Brca2. These observations suggest that the gender-specific differences in phenotype associated with germline mutations in BRCA2 versus BRCA1 may be related to the differential regulation of these genes by sex hormones.

Brca2 is coordinately regulated with Brca1 during proliferation and differentiation in mammary epithelial cells.

We have analyzed the expression of the breast cancer susceptibility gene, Brca2, in mammary epithelial cells as a function of proliferation and differentiation. Our results demonstrate that Brca2 mRNA expression is tightly regulated during mammary epithelial proliferation and differentiation, and that this regulation occurs coordinately with Brca1. Specifically, Brca2 mRNA expression is up-regulated in rapidly proliferating cells; is down-regulated in response to serum deprivation; is expressed in a cell cycle-dependent manner, peaking at the G1/S boundary; and is up-regulated in differentiating mammary epithelial cells in response to glucocorticoids. In each case, an identical pattern of expression was observed for Brca1. These results indicate that proliferative stimuli modulate the mRNA expression of these two breast cancer susceptibility genes. In addition, the coordinate regulation of Brca1 and Brca2 revealed by these experiments suggests that these genes are induced by, and may function in, overlapping regulatory pathways involved in the control of cell proliferation and differentiation.