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Biomedical subjects

D F Stern

Publications and source records attributed to D F Stern.

At least 19 recordsLinked to original sources
PubMed2006

Formation of Neu/ErbB2-induced mammary tumors is unaffected by loss of ErbB4.

The four members of the ErbB family of receptor tyrosine kinases are involved in development and tumorigenesis of the mammary gland. Whereas the epidermal growth factor receptor, ErbB2 and ErbB3 are positively associated with various cancers, clinical studies of ErbB4 in breast cancer are contradictory. Results from tissue culture analyses and some clinical studies suggested that ErbB4 is either a tumor suppressor or is a negative regulator of ErbB2-driven tumors. Neu-Cre-ErbB4(flox/null) mice in which ErbB4 was inactivated by Cre-lox-mediated recombination in the mammary gland developed MMTV-Neu-driven mammary tumors with a similar latency period to mice with one or two wild-type ErbB4 alleles. Moreover, there was no difference in the histologies of tumors that developed, nor in the propensity to form lung metastases. Taken together these results suggest that ErbB4 is not a potent, highly penetrant tumor suppressor, nor is it a factor in Neu-mediated tumorigenesis in this model.

Animals↗
PubMed2001

Pathobiologic findings in DCIS of the breast: morphologic features, angiogenesis, HER-2/neu and hormone receptors.

With the increasing incidence of ductal carcinoma in situ (DCIS) of the breast and its relationship to invasive breast carcinoma, it is important to understand the biology of this entity. We report on a hospital-based survey of 219 case subjects with DCIS of the breast without associated invasive carcinoma diagnosed between 1982 and 1994. The cases of DCIS were analyzed for architectural type, size, nuclear grade, necrosis, calcification, periductal fibrosis, neovascularization, estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu expression. Periductal neovascularization was associated with tumor size, microcalcifications, periductal fibrosis, and HER-2/neu overexpression. Expression of ER and PR was observed in 60 and 62% of the cases, respectively, and HER-2/neu overexpression in 28% of the cases. ER and PR expression were both inversely associated with comedo histology and nuclear grade. HER-2/neu overexpression was positively associated with comedo histology, high nuclear grade, and periductal neovascularization and was inversely correlated with both ER and PR expression. High nuclear grade was positively associated with comedocarcinoma, necrosis, microcalcification, and periductal fibrosis. The role of these molecular/pathologic markers in the biology of DCIS and their potential clinical implications are discussed.

Breast Neoplasms↗
PubMed2000

Activation (tyrosine phosphorylation) of ErbB-2 (HER-2/neu): a study of incidence and correlation with outcome in breast cancer.

PURPOSE: We hypothesize that phosphorylated ErbB-2 (P-ErbB-2, identified by a novel antibody PN2A) may provide either more significant or additional prognostic marker data for breast cancer patients. This study was designed to compare the incidence and prognostic value of ErbB-2 (HER-2/neu) and P-ErbB-2 immunoexpression in archival breast cancer samples. MATERIALS AND METHODS: Eight hundred sixteen invasive breast cancers with a median of 16.3 years of follow-up were immunostained for ErbB-2 (using antibody CB11) and P-ErbB-2 (using antibody PN2A). ErbB-2 and P-ErbB-2 data were compared with clinical, histologic, immunohistochemical, and outcome variables. RESULTS: Of 816 primary breast cancers, 307 (38%) were positive for ErbB-2 and 37 (12% of ErbB-2 positive and 5% of the study population) expressed P-ErbB-2. P-ErbB-2 was not detected in ErbB-2-negative cases (n = 509). ErbB-2 immunohistochemical data were bimodal; patients with > or = 80% cellular expression had the shortest disease-free and disease-specific survival. P-ErbB-2 was associated with a higher percentage of ErbB-2-positive cells, a higher number of positive lymph nodes, and cellular proliferation. ErbB-2 and P-ErbB-2 were indicators of poor prognosis in node-positive patients in both univariate and multivariate analyses. We found that either P-ErbB-2 expression or high (> or = 80%) ErbB-2 expression provided the most significant prognostic value in node-positive cases by multivariate analyses. There were too few P-ErbB-2-positive cases and events in the node-negative patient group to allow statistical analysis of P-ErbB-2 in that subgroup. CONCLUSION: PN2A immunostaining identified a subset (approximately 12% of ErbB-2-positive breast cancers) with activation (phosphorylation) of the receptor ErbB-2. P-ErbB-2 expression was strongly associated with higher levels of ErbB-2 expression (> or = 80%), although it was not a surrogate. Identification of cases with a high percentage of invasive breast cancer cells expressing ErbB-2 or determination of receptor activation via P-ErbB-2 may provide additional prognostic value in node-positive breast cancers.

Adult↗
PubMed2000

Tyrosine kinase signalling in breast cancer: ErbB family receptor tyrosine kinases.

ERBB family receptor tyrosine kinases are overexpressed in a significant subset of breast cancers. One of these receptors, HER2/neu, or ErbB-2, is the target for a new rational therapeutic antibody, Herceptin. Other inhibitors that target this receptor, and another family member, the epidermal growth factor (EGF) receptor, are moving into clinical trials. Both of these receptors are sometimes overexpressed in breast cancer, and still subject to regulation by hormones and other physiological regulators. Optimal use of therapeutics targeting these receptors will require consideration of the several modes of regulation of these receptors and their interactions with steroid receptors.

Antibodies, Monoclonal↗
PubMed1999

ErbB4 signaling in the mammary gland is required for lobuloalveolar development and Stat5 activation during lactation.

Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4DeltaIC) as a transgene in the mammary gland. Despite the expression of ErbB4DeltaIC from puberty through later stages of mammary development, an ErbB4DeltaIC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4DeltaIC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, beta-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and alpha-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4DeltaIC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.

Animals↗
PubMed1999

Expression of dominant-negative ErbB2 in the mammary gland of transgenic mice reveals a role in lobuloalveolar development and lactation.

Overexpression of the receptor tyrosine kinase ErbB2/HER2/Neu (ErbB2) occurs in 15-40% of human breast cancers. To determine the function of ErbB2 signaling during normal mouse mammary gland development, we expressed a carboxyl-terminal truncated dominant negative allele of ErbB2 (ErbB2deltaIC) in the developing mouse mammary gland. Despite ErbB2deltaIC expression within mammary glands of pubescent virgin and pregnant mice, a phenotype was not observed until late in pregnancy. At 1 day post-partum, lactationally active, distended lobuloalveoli failed to form. This phenotype was exaggerated in multiparous females expressing ErbB2deltaIC. Immunohistochemical staining for ErbB2deltaIC revealed a concordance between high levels of ErbB2deltaIC protein expression and the absence of lactational products within the lumens of ErbB2deltaIC stained lobuloalveoli. These results demonstrate that ErbB2 signaling is required for proper mammary development and lactation at parturition.

3T3 Cells↗
PubMed1999

Neuregulin activation of ErbB receptors in vascular endothelium leads to angiogenesis.

The ErbB, or epidermal growth factor receptor (EGF-r), family of transmembrane tyrosine kinase receptors has been demonstrated to play an important role in growth regulation and intracellular signaling in a wide variety of cell types. Targeted deletion of neuregulin (an ErbB ligand) in mice results in endocardial cushion abnormalities, suggesting that these receptor-ligand interactions have important effects on vascular endothelial growth and development. To study the role of ErbB receptor signaling in vascular endothelium, we investigated the expression pattern of the various receptor family members and the effect of ErbB receptor stimulation in human umbilical vein endothelial cells (HUVEC). We demonstrate that ErbB2 (neu), ErbB3, and ErbB4 are highly expressed, whereas ErbB1 (EGF-r) is undetectable. Stimulation of HUVEC with recombinant neuregulin-beta (an ErbB3/4 ligand) induces rapid calcium fluxes, receptor tyrosine phosphorylation, and cell proliferation. We demonstrate marked in vitro and in vivo angiogenic responses to neuregulin-beta, which are independent of vascular endothelial cell growth factor. These findings support an important role for the ErbB family of receptors in endothelial cell signaling and function, including neuregulin-induced angiogenesis.

Animals↗
PubMed1998

Active signaling by Neu in transgenic mice.

Transgenic mice engineered to overexpress the HER-2/neu/erbB-2 protooncogene under the control of a mammary-specific promoter develop mammary tumors and are a model for human breast cancer. Signal transduction by Neu was examined in situ in the tumors of these transgenic mice. This was accomplished using the PN2A monoclonal antibody, which recognizes Neu only in the phosphorylated, and therefore actively signaling, state. Immunohistochemistry using PN2A demonstrated that Neu actively signals in the tumors of Neu transgenic mice. Expression of Neu was always accompanied by co-overexpression of the endogenous epidermal growth factor receptor. Qualitatively similar results were found in mammary tumors from mice bitransgenic for the neu and transforming growth factor-alpha genes (both driven by the mouse mammary tumor virus promoter). Early mammary lesions demonstrated distinctive patterns of Neu activation relative to expression levels. Overexpression and activation were separable both temporally and spatially. These results refine the multi-step model for the role of Neu in mammary neoplasia and establish phosphorylation-state specific antibodies as a powerful tool for investigating tumor progression.

Animals↗
PubMed1998

Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint.

The Rad53 protein kinase of Saccharomyces cerevisiae is required for checkpoints that prevent cell division in cells with damaged or incompletely replicated DNA. The Rad9 protein was phosphorylated in response to DNA damage, and phosphorylated Rad9 interacted with the COOH-terminal forkhead homology-associated (FHA) domain of Rad53. Inactivation of this domain abolished DNA damage-dependent Rad53 phosphorylation, G2/M cell cycle phase arrest, and increase of RNR3 transcription but did not affect replication inhibition-dependent Rad53 phosphorylation. Thus, Rad53 integrates DNA damage signals by coupling with phosphorylated Rad9. The hitherto uncharacterized FHA domain appears to be a modular protein-binding domain.

Amino Acid Sequence↗
PubMed1998

Activation of ErbB4 by the bifunctional epidermal growth factor family hormone epiregulin is regulated by ErbB2.

Epiregulin (EPR) is a recently described member of the epidermal growth factor (EGF) family of peptide growth factors. The ever expanding size of the EGF family has made distinguishing the activities of these hormones paramount. We show here that EPR activates two members of the ErbB family of receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and ErbB4. Therefore by these criteria, EPR is qualitatively similar to another EGF family hormone, betacellulin (BTC). Yet, here we also demonstrate quantitative differences between EPR and BTC. EPR stimulates higher levels of EGFR phosphorylation than does BTC, whereas BTC stimulates higher levels of ErbB4 phosphorylation than does EPR. Moreover, the EPR and BTC dose response curves show that although EGFR is more sensitive to EPR than is ErbB4, ErbB4 is more sensitive to BTC than is EGFR. Finally, ErbB2, which is not activated by EPR when expressed on its own, increases the sensitivity of ErbB4 for activation by EPR. Therefore, these results establish that EPR exhibits novel activities and modes of regulation, which may have significant implications for EPR function in vivo.

Animals↗
PubMed1998

Specificity within the EGF family/ErbB receptor family signaling network.

Recent years have witnessed tremendous growth in the epidermal growth factor (EGF) family of peptide growth factors and the ErbB family of tyrosine kinases, the receptors for these factors. Accompanying this growth has been an increased appreciation for the roles these molecules play in tumorigenesis and in regulating cell proliferation and differentiation during development. Consequently, a significant question has been how diverse biological responses are specified by these hormones and receptors. Here we discuss several characteristics of hormone-receptor interactions and receptor coupling that contribute to specificity: 1) a single EGF family hormone can bind multiple receptors; 2) a single ErbB family receptor can bind multiple hormones; 3) there are three distinct functional groups of EGF family hormones; 4) EGF family hormones can activate receptors in trans, and this heterodimerization diversifies biological responses; 5) ErbB3 requires a receptor partner for signaling; and 6) ErbB family receptors differentially couple to signaling pathways and biological responses.

Animals↗
PubMed1998

Activation of Neu (ErbB-2) mediated by disulfide bond-induced dimerization reveals a receptor tyrosine kinase dimer interface.

Receptor dimerization is a crucial intermediate step in activation of signaling by receptor tyrosine kinases (RTKs). However, dimerization of the RTK Neu (also designated ErbB-2, HER-2, and p185(neu)), while necessary, is not sufficient for signaling. Earlier work in our laboratory had shown that introduction of an ectopic cysteine into the Neu juxtamembrane domain induces Neu dimerization but not signaling. Since Neu signaling does require dimerization, we hypothesized that there are additional constraints that govern signaling ability. With the importance of the interreceptor cross-phosphorylation reaction, a likely constraint was the relative geometry of receptors within the dimer. We have tested this possibility by constructing a consecutive series of cysteine substitutions in the Neu juxtamembrane domain in order to force dimerization along a series of interreceptor faces. Within the group that dimerized constitutively, a subset had transforming activity. The substitutions in this subset all mapped to the same face of a predicted alpha helix, the most likely conformation for the intramembrane domain. Furthermore, this face of interaction aligns with the projected Neu* V664E substitution and with a predicted amphipathic interface in the Neu juxtamembrane domain. We propose that these results identify an RTK dimer interface and that dimerization of this RTK induces an extended contact between juxtamembrane and intramembrane alpha helices.

3T3 Cells↗
PubMed1997

Ligands for ErbB-family receptors encoded by a neuregulin-like gene.

Neuregulins (also called ARIA, GGF, heregulin or NDF) are a group of polypeptide factors that arise from alternative RNA splicing of a single gene. Through their interaction with the ErbB family of receptors (ErbB2, ErbB3 and ErbB4), neuregulins help to regulate cell growth and differentiation in many tissues. Here we report the cloning of a second neuregulin-like gene, neuregulin-2. The encoded product of the neuregulin-2 gene has a motif structure similar to that of neuregulins and an alternative splicing site in the epidermal growth factor(EGF)-like domain gives rise to two isoforms (alpha and beta). Northern blot and in situ hybridization analysis of adult rat tissues indicate that expression of neuregulin-2 is highest in the cerebellum, and the expression pattern is different from that of neuregulins. Recombinant neuregulin-2beta induces the tyrosine-phosphorylation of ErbB2, ErbB3 and ErbB4 in cell lines expressing all of these ErbB-family receptors. However, in cell lines with defined combinations of ErbBs, neuregulin-2beta only activates those with ErbB3 and/or ErbB4, suggesting that signalling by neuregulin-2 is mediated by ErbB3 and/or ErbB4 receptors.

Amino Acid Sequence↗
PubMed1997

Dimerization of the p185neu transmembrane domain is necessary but not sufficient for transformation.

The neu proto-oncogene encodes a receptor tyrosine kinase (RTK). The oncogenic allele neu* (p185*) bears a glutamic acid for valine substitution at position 664 within the predicted transmembrane domain. We have used this mutant to explore the role of the transmembrane domain in signal transduction by RTKs. Analysis of a panel of neu* proteins with second-site mutations in the transmembrane domain revealed a strong correlation of dimerization with transformation. Both dimerization and transformation are dependent on a domain formed by the amino acids Val663-Glu664-Gly665 (VEG). However, movement of the VEG elsewhere within the transmembrane domain promoted weak dimerization but not transformation. Epidermal growth factor receptor (EGFR)/neu chimeras were used to determine if mutations that disrupt activation by Glu664 affect hormone-regulated signal transduction as well. These mutations (of Val663 and Gly665) did not affect regulation by EGF. Introduction of the known transmembrane dimerization domain from Glycophorin A (GpA) stimulated dimerization, but was not sufficient for transformation. These results indicate that dimerization is necessary but not sufficient for transforming activity. The homologous wild-type domain, VVG, is not required for hormone-regulated signaling.

3T3 Cells↗
PubMed1997

Cripto enhances the tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase (MAPK) in mammary epithelial cells.

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor alpha, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin beta1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42(erk-2) mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Animals↗
PubMed1997

A role for DNA primase in coupling DNA replication to DNA damage response.

The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.

Blotting, Western↗
PubMed1997

Mutations in SPK1/RAD53 that specifically abolish checkpoint but not growth-related functions.

SPK1/RAD53/SAD1/MEC2 encodes an essential protein kinase of Saccharomyces cerevisiae and is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We have isolated two mutant alleles of SPK1 (spk1K227A and spk1-1A208P) that are defective for checkpoint-arrest functions but retain wild-type levels of SPK1-associated growth activity. Both mutations occur within conserved regions of the kinase domain of SPK1 resulting in a substantial reduction in the catalytic activity of Spk1. Thus, while minimal levels of Spk1 kinase activity are capable of supporting normal rates of growth, higher levels are required for checkpoint functions. In addition, using deletional analysis we have identified a region within the N-terminus of Spk1 outside of the conserved kinase domain that is required for checkpoint functions. Interestingly, this region may be important in the regulation of Spk1 kinase activity.

Alleles↗