Prolactin-Inducible Protein: From Breast Cancer Biomarker to Immune Modulator-Novel Insights from Knockout Mice.

The propensity for breast cancers to elicit immune responses in patients is well established. The accumulation of tumor infiltrating lymphocytes within the primary breast tumor has been linked to better prognosis and better response to therapy. The prolactin-inducible protein (PIP) is a 15 kD protein that is expressed under physiological conditions of the breast and is regarded as a marker of mammary differentiation. While highly expressed under pathological conditions of the mammary gland, including breast cancers, PIP is expressed in very few other cancers. Although the function of PIP is not well elucidated, numerous studies suggest that its primary role may be related to host defense and immune modulation. However, evidence to show a direct link between PIP and the immune response has been lacking. In this review, we discuss our recent work with Pip-deficient mice, linking PIP not only to a role in innate immunity but for the first time, providing evidence for a role in cell-mediated immunity. These functional studies in Pip null mice lend new insight into the role of PIP in immunity and suggest that PIP may play a similar immune-regulatory role in breast cancer.

Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to adriamycin.

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21(waf1/cip1) and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21(waf1/cip1), down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.

Targeted production of proprotein convertase PC1 enhances mammary development and tumorigenesis in transgenic mice.

Elevated production of proprotein convertases (PCs), proteolytic enzymes that posttranslationally modify the biological activities of diverse groups of cellular proteins, is a common occurrence in human breast carcinomas. A transgenic mouse model was developed to gain insight into the significance of PC production in breast development and neoplasia. Mammary epithelium-specific and early expression of PC1 was targeted by the use of the mouse mammary tumor virus promoter/enhancer. Whole-mount examinations revealed that the mammary glands of 83-day-old virgin PC1 transgenic mice exhibited an accelerated lobuloalveolar development compared with that of age-matched wild-type mice (p < 0.001). This phenotypic change was accompanied by extensive alterations in gene expression assessed by gene expression microarray analyses. Pathway analysis of PC1-induced alterations in gene expression has revealed possible mechanism of action of PC1 in the mammary gland. PC1 expression alone, however, did not promote spontaneous mammary tumorigenesis in the transgenic mice. PC1 transgene expression resulted in a significantly higher incidence (p = 0.008) and accelerated growth (p = 0.023) of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas. The present study therefore shows that PC1 expression can promote normal and neoplastic mammary development and growth and suggests that proprotein convertases may be important etiological factors in human breast neoplasia.

Cells deficient in oxidative DNA damage repair genes Myh and Ogg1 are sensitive to oxidants with increased G2/M arrest and multinucleation.

Oxidative stress generated from endogenous and exogenous sources causes oxidative DNA damage. The most frequent mutagenic base lesion 7,8-dihydro-8-oxoguanine and the resulting mismatched adenine are removed by OGG1 and MYH in mammals. Deficiencies in human MYH or mouse MYH and OGG1 result in tumor predisposition but the underlying molecular mechanism is not fully understood. To facilitate the study of the roles of MYH and OGG1 in the protection against oxidative stress, we generated mouse embryonic fibroblast cell lines deficient in these genes. Myh and Ogg1 double knockout cells were more sensitive than wild type to oxidants (hydrogen peroxide and t-butyl hydroperoxide), but not to cis-platinum or gamma-irradiations. The low dosage oxidative stress resulted in more reduction of S phase and increase of G(2)/M phase in Myh(-/-)Ogg1(-/-) cells than in wild-type cells, but a similar level of cell death in both cells. The oxidants also induced more multinucleated cells in Myh(-/-)Ogg1(-/-) cells than in wild-type, accompanied by centrosome amplification and multipolar spindle formation. Thus, under oxidative stress, Myh and Ogg1 are likely required for normal cell-cycle progression and nuclear division, suggesting multiple roles of Myh and Ogg1 in the maintenance of genome stability and tumor prevention.

A real-time PCR method for the quantification of the two isoforms of metallothionein in Lake Trout (Salvelinus namaycush).

Metallothioneins (MTs) are low-molecular-weight proteins whose physiologic roles are the regulation of essential metals Cu and Zn, sequestration of heavy metals, and free radical scavenging. Induced production of MTs in a wide variety of organisms exposed to heavy metals has made them popular exposure indicators. While it has been postulated that the three different isoforms of MT play different physiologic roles, methods to discern induction separately have not been available. The development of real-time polymerase chain reaction (real-time PCR) primers and TaqMan probes to measure the two MT isoforms found in salmonid fish are described. Assuming a high degree of homology between the isoforms and within different groups of salmonids, the sequences for MT-I and MT-II from rainbow trout were used to develop primers and probes for lake trout using the Primer3 program. Two sections of each isoform that varied by only a few nucleotides were targeted. SYBR Green validated the primer specificity, and melt curve analysis further ensured that only one product was amplified. Analysis of archived samples from fish captured in unmanipulated reference lakes or from lakes experimentally treated with cadmium or ethynylestradiol (EE2) afforded an examination of seasonal and contaminant influences on MT-I and MT-II mRNA expression.

Gene expression profiling of early involuting mammary gland reveals novel genes potentially relevant to human breast cancer.

Mammary gland involution represents one of the most dramatic examples of programmed cell death/apoptosis and tissue regression during development, yet large gaps still exist in the understanding of the mechanisms involved, and the key factors that trigger involution, are not yet identified. With the focus on identifying "novel" genes associated with mammary gland regression, we used microarray analysis to examine differentially expressed genes during early mammary gland involution in the mouse. We then examined the relevance of candidate genes to human tumorigenesis and identified a number of genes not previously implicated or not well characterized in human breast cancer. The expression levels of these genes in human breast cancer were confirmed in breast cancer cell lines and breast tumor tissues. This pilot study demonstrates proof of principle that through the analysis of gene expression during mammary gland involution, it may be possible to identify "novel" genes relevant human breast cancer.

Estrogen receptor-beta regulates psoriasin (S100A7) in human breast cancer.

We have previously observed a paradoxical relationship of the psoriasin/S100A7 gene with estrogen response in-vitro in ERalpha positive cells but its association with ERalpha negative status in-vivo raising the possibility that S100A7 might be regulated by ERbeta in breast cancer. Using doxycycline-inducible ERbeta and ERalpha expressing MCF-7 cells the hypothesis that psoriasin/S100A7 is ERbeta regulated was investigated To explore the relationship between psoriasin/S100A7 and ERbeta expression in-vivo, we also assessed a cohort of 233 ERalpha negative breast tumors using tissue microarrays and immunohistochemistry. Psoriasin/S100A7 was increased by 17beta-estradiol (E2) following ERbeta induction, in several clones of ERbeta over-expressing but not in the original MCF-7 cells, nor clones over-expressing ERalpha. The effect of E2 on psoriasin/S100A7 was inhibited by 4-hydroxytamoxifen and ICI 182780 but not with a selective ERalpha antagonist. An ERbeta selective-agonist but not an ERalpha selective-agonist, induced psoriasin/S100A7. This induction still occurred after stable down-regulation of ERalpha using siRNA in ERbeta inducible cells. E2 increased psoriasin/S100A7 mRNA but cycloheximide treatment inhibited this effect. A relationship between ERbeta and psoriasin/S100A7 was observed in the p53 immunohistochemically negative subset of invasive breast tumors in-vivo (r = 0.225, p = 0.046, n = 79). In conclusion we demonstrate that E2 induction of psoriasin/S100A7 can be specifically regulated through ERbeta in-vitro and associated with ERbeta in-vivo. These data support the hypothesis that psoriasin/S100A7 is specifically regulated by ERbeta activity and could be useful to guide future therapies targeting ERbeta in certain phenotypic subsets of breast cancers in-vivo.

Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery.

1,25-Dihydroxyvitamin D(3) and vitamin D(3) analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-l-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiation-induced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse.

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.

Expression analysis of the mouse S100A7/psoriasin gene in skin inflammation and mammary tumorigenesis.

BACKGROUND: The human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis. METHODS: On the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7. RESULTS: Although mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene. CONCLUSION: These observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression.

Developmental changes in insulin-like growth factor I receptor gene expression in the mouse mammary gland.

The insulin-like growth factor I receptor (IGF-IR), which mediates the mitogenic action of IGF-I, has been shown to play an essential role in normal growth and development. However, the precise role of IGF-IR in the growth and differentiation of the mammary gland has not been elucidated. This study examines the profile of the IGF-IR gene and protein expression during normal postnatal mammary gland development in order to gain further insight into the role of the IGF-I/IGF-IR during mammary gland morphogenesis. Gene and protein expression were examined in developing mouse mammary glands (virgin, pregnant, lactating, involuting) by real time PCR analysis and Western blotting. Both IGF-IR gene and protein expression levels were high during early pregnancy. Interestingly, the level of gene expression was significantly down-regulated during late pregnancy (5.4 fold) and lactation (9-13 fold) and significantly up-regulated (3.9 fold) during late involution, to the level observed in the virgin mammary gland. By in situ hybridization, the IGF-IR transcripts were localized to the proliferating ductal epithelium of the mammary glands of virgin mice and to the differentiating ductal and alveolar epithelium of the mammary glands during pregnancy and lactation. In the involuting gland, the transcripts were localized to the regressing ductal epithelium. These data are direct evidence that IGF-IR expression is important for alveolar cell proliferation and suggest that the progression of involution may require the down-regulation of IGF-IR gene expression. Altogether, these results demonstrate that a developmental IGF-IR gene expression pattern exists in the mouse mammary gland and that increases in gene expression at specific phases of development may reflect an important role for IGF-I/IGF-IR at those phases of development.

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.

C-myc gene expression alone is sufficient to confer resistance to antiestrogen in human breast cancer cells.

C-myc is implicated in the initiation, progression and estrogen response of breast cancer. To further investigate the role of c-myc in breast cancer, we have developed clonal MCF-7 human breast cancer cell lines harboring a stably-transfected human c-myc gene, whose expression was stringently controlled by the bacterial reverse tetracycline transcription activator protein. The expression of the endogenous genomic c-myc gene in MCF-7 cells was abolished by the potent pure estrogen antagonist, ICI 182,780. Functional c-Myc protein was identified by both Western immunoblotting and by its ability to transactivate a chimeric plasmid consisting of E-box sequences upstream of the luciferase reporter gene. One MCF-7 clone, 35im, was chosen for further characterization. C-myc induction by doxycycline was rapid and dose dependent; c-myc mRNA appeared as early as 30 min after doxycycline addition and stimulation of c-myc expression required as little as 50 ng/ml doxycycline, with c-myc mRNA levels reaching a plateau at 2.5 microg/ml doxycycline. ICI 182,780 or doxycycline (a tetracycline analog) treatment did not alter the mRNA levels of Max, the c-myc binding partner. As in wildtype MCF-7 cells, the growth of clone 35im was inhibited by 1 microM or less of ICI 182,780 and stimulated by 10 nM to 1 microM 17beta-estradiol. When maintained in a complete medium containing 5% normal fetal bovine serum (FBS) and ICI 182,780, doxycycline induced cell growth by 400% in an 8-day assay. A similar level of growth was achieved with doxycycline treatment in cells that were arrested by the use of charcoal-stripped FBS. Doxycycline had no effect on the growth of a control MCF-7 clone (18c). Apoptosis, assessed by caspase-dependent cleavage of poly(ADP-ribose) polymerase, was unchanged in clone 35im cells after treatments with doxycycline or ICI 182,780. The present study demonstrates that c-myc alone is sufficient to confer antiestrogen resistance in human breast cancer. Our novel c-myc-inducible MCF-7 cell model offers a unique opportunity to study the diverse actions of the c-myc proto-oncogene in human breast cancer.