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.

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.

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.

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.

Cloning, characterization, and chromosomal localization of Pnck, a Ca(2+)/calmodulin-dependent protein kinase.

Calcium is an important second messenger in eukaryotic cells. Many of the effects of calcium are mediated via its interaction with calmodulin and the subsequent activation of Ca(2+)/calmodulin-dependent (CaM) kinases. CaM kinases are involved in a wide variety of cellular processes including muscle contraction, neurotransmitter release, cell cycle control, and transcriptional regulation. While CaMKII has been implicated in learning and memory, the biological role of the other multifunctional CaM kinases, CaMKI and CaMKIV, is largely unknown. In the course of a degenerate RT-PCR protein kinase screen, we identified a novel serine/threonine kinase, Pnck. In this report, we describe the cloning, chromosomal localization, and expression of Pnck, which encodes a 38-kDa protein kinase whose catalytic domain shares 45-70% identity with members of the CaM kinase family. The gene for Pnck localizes to mouse chromosome X, in a region of conserved synteny with human chromosome Xq28 that is associated with multiple distinct mental retardation syndromes. Pnck is upregulated during intermediate and late stages of murine fetal development with highest levels of expression in developing brain, bone, and gut. Pnck is also expressed in a tissue-specific manner in adult mice with highest levels of expression detected in brain, uterus, ovary, and testis. Interestingly, Pnck expression in these tissues is restricted to particular compartments and appears to be further restricted to subsets of cells within those compartments. The chromosomal localization of Pnck, along with its tissue-specific and restricted pattern of spatial expression during development, suggests that Pnck may be involved in a variety of developmental processes including development of the central nervous system.

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.

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.

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.