Gail M. Clinton

p95HER-2 Predicts Worse Outcome in Patients with HER-2-Positive Breast Cancer

Background: The HER-2 receptor undergoes a proteolytic cleavage generating an NH2-terminally truncated fragment, p95HER-2, that is membrane-associated and tyrosine-phosphorylated. We have reported that p95HER-2, but not the full-length receptor, p185HER-2, correlated with the extent of lymph node involvement in patients with breast cancer and its expression was significantly enhanced in nodal metastatic tissue. These facts suggested an important role for p95HER-2 either as a marker or cause of metastasis and poor outcome in breast cancer. In this work, we have studied the prognostic value of p95HER-2 in breast cancer. Methods: Primary breast tumor tissues (n = 483) were from surgical resections conducted in hospitals in two different countries: the U.S. (n = 334) and Spain (n = 149). HER-2 protein forms, including p185HER-2 and p95HER-2, were examined in extracts of primary breast tumors by Western blot analysis. The levels of the two forms (high or low) were tested for association with other clinicopathologic factors and for correlation with disease-free survival. Results: The median follow-up was 46 months. A high level of p95HER-2 in primary tumor tissue correlated with reduced 5-year disease-free survival (hazard ratio, 2.55; 95% confidence interval, 2.13-8.01; P < 0.0001). The median time for disease-free survival was 32 versus 139 months in patients with low levels of p95HER-2. In comparison, high levels of the full-length p185HER-2 did not significantly correlate with poor outcome (P > 0.1). Multivariate analysis revealed that high p95HER-2 was an independent predictor of disease-free survival (hazard ratio, 1.59; 95% confidence interval, 1.246-1.990; P = 0.0004). Conclusions: p95HER-2 expression is an independent prognostic factor in breast cancer and defines a group of patients with HER-2-positive breast cancer with significantly worse outcome.

Estrogen action in human ovarian cancer.

Evidence is accumulating for a facilitative role for estrogen in ovarian cancer. Although response to antiestrogen therapy has been poor, there is a distinct subset of patients that respond. Strategies for treatment of ovarian cancer would be improved by identification of patients likely to respond to hormonal therapy. Cell culture models that are responsive or resistant to estrogen and antiestrogen may be of value in finding markers that predict responsiveness to hormonal therapy. Several model cell lines have been generated that express ER and proliferate in response to estrogen in vitro. Further studies are needed to better characterize the response of these ER positive cells lines to estrogen in vivo in mouse xenograft models. Expression of many of the same genes are regulated by estrogen in breast and in ovarian cancer cell lines. One exception may be the HER-2/neu oncogene product, which is down-regulated by estrogen in responsive breast carcinoma cells but not in two ovarian carcinoma cell lines. Initial analyses of several estrogen responsive and one resistant cell model suggests the potential value of progesterone receptor presence and low levels of HER-2/neu expression for predicting responsiveness to hormonal therapy. Additional cell models need to be investigated to determine the frequency with which these markers are associated with antiestrogen resistance.

SKOV3 ovarian carcinoma cells have functional estrogen receptor but are growth-resistant to estrogen and antiestrogens

Estrogen receptor positive ovarian cancer is often refractile to antiestrogen therapy. Here we describe the SKOV3 human ovarian carcinoma cell line as an in vitro model for estrogen and antiestrogen resistant ovarian cancer. While SKOV3 cells expressed estrogen receptor (ER) mRNA and protein at a similar level as the estrogen responsive T47D breast carcinoma cell line, their growth was not responsive to estradiol (E2) and was not inhibited by the antiestrogens OH-tamoxifen and ICI 164,384. The ER in SKOV3 cells was normal with respect to apparent Kd for binding with E2, E2 regulation of a transiently transfected ERE driven reporter gene, and E2 stimulation of expression of the early growth response genes c-myc and c-fos. However, the SKOV3 cells exhibited no expression of the progesterone receptor gene (PR) even after addition of E2, and the protein products of the estrogen responsive genes HER-2/neu and cathepsin D were expressed at constitutive levels that were not regulated by E2. Therefore, estrogen resistance in these cells may be a result of constitutive expression and loss of E2 regulation of selected growth regulatory gene products rather than a defect in estrogen activation of ER as a transcriptional regulator.

Expression of herstatin, an autoinhibitor of HER-2/neu, inhibits transactivation of HER-3 by HER-2 and blocks EGF activation of the EGF receptor.

The four members of the EGF receptor family are capable of homomeric as well as heteromeric interactions. HER-2/neu (erbB-2) dominates as the preferred coreceptor that amplifies mitogenic signaling. An alternative HER-2/neu product, herstatin, consists of a segment of the ectodomain of p185HER-2 and an intron-encoded C-terminus. Recombinant herstatin was found to bind with nM affinity and inhibit p185HER-2. To further examine the impact on receptor activity, herstatin was expressed with various receptor tyrosine kinases. In CHO cells that overexpressed HER-2, herstatin caused a sevenfold inhibition of colony formation that corresponded to a reduction in the tyrosine phosphorylation of p185HER-2. Herstatin also prevented HER-2 mediated transactivation of the kinase impaired HER-3 as reflected in transphosphorylation of HER-3 and heteromers between HER-2 and HER-3. In EGF receptor-overexpressing cells, EGF induction of receptor dimerization and tyrosine phosphorylation were reduced more than 90%, and receptor down-regulation as well as colony formation were also suppressed by coexpression with herstatin. Inhibition was selective for the EGF receptor family since herstatin expression did not reduce tyrosine phosphorylation mediated by the FGF receptor-2 or by insulin-like growth factor -1. Herstatin bound to the EGF receptor as well as to p185HER-2 in pull-down assays suggesting that complex formation may be involved in receptor inhibition. Our findings indicate that herstatin has the capability to negatively regulate combinations of interactions between group I receptor tyrosine kinases that confer synergistic growth signals.

The epidermal growth factor receptor from prostate cells is dephosphorylated by a prostate-specific phosphotyrosyl phosphatase

Human prostatic acid phosphatase (PAcP) has been found to have phosphotyrosyl-protein phosphatase activity (H. C. Li, J. Chernoff, L. B. Chen, and A. Kirschonbaun, Eur. J. Biochem. 138:45-51, 1984; M.-F. Lin and G. M. Clinton, Biochem. J. 235:351-357, 1986) and has been suggested to negatively regulate phosphotyrosine levels, at least in part, by inhibition of tyrosine protein kinase activity (M.-F. Lin and G. M. Clinton, Adv. Protein Phosphatases 4:199-228, 1987; M.-F. Lin, C. L. Lee, and G. M. Clinton, Mol. Cell. Biol. 6:4753-4757, 1986). We investigated the molecular interaction of PAcP with a specific tyrosine kinase, the epidermal growth factor (EGF) receptor, from prostate carcinoma cells. Of several proteins phosphorylated in membrane vesicles from prostate carcinoma cells, PAcP selectively dephosphorylated the EGF receptor. The prostate EGF receptor was more efficiently dephosphorylated by PAcP than by another phosphotyrosyl phosphatase, potato acid phosphatase. Further characterization of the interaction of PAcP with the EGF receptor revealed that the optimal rate of dephosphorylation occurred at neutral rather than at acid pH. Thus, the enzyme that we formerly referred to as PAcP we now call prostatic phosphotyrosyl-protein phosphatase. Hydrolysis of phosphate from tyrosine residues in the immunoprecipitated EGF receptor catalyzed by purified prostatic phosphotyrosyl-protein phosphatase caused a 40 to 50% decrease in the receptor tyrosine kinase activity with angiotensin as the substrate. In contrast, autophosphorylation of the receptor was associated with an increase in tyrosine kinase activity.

Herstatin, an Autoinhibitor of the Human Epidermal Growth Factor Receptor 2 Tyrosine Kinase, Modulates Epidermal Growth Factor Signaling Pathways Resulting in Growth Arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor α (TGF-α) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-α, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-α induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-α-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-α signaling profiles, culminating in inhibition of proliferation.

Herstatin inhibits heregulin-mediated breast cancer cell growth and overcomes tamoxifen resistance in breast cancer cells that overexpress HER-2.

Ligands of the ErbB family of receptors and estrogens control the proliferation of breast cancer cells. Overexpression of human EGF receptor HER-2 (erbB2) leads to amplified heregulin (HRG) signaling, promoting more aggressive breast cancer that is nonresponsive to estrogen and the antiestrogenic drug tamoxifen. Herstatin (Hst), a secreted HER-2 gene product, binds to the HER-2 receptor ectodomain blocking receptor activation. The aim of this study was to investigate the impact of this HER-2 inhibitor on HRG-induced signaling, proliferation, and sensitivity to tamoxifen in breast cancer cells with and without HER-2 overexpression. The expression of Hst in MCF7 cells eliminated HRG signaling through both mitogen-activated protein kinase and Akt pathways and prevented HRG-mediated proliferation. The loss in signaling corresponded to downregulation of the HRG receptors, HER-3 and HER-4, whereas HER-2 overexpression strongly stimulated the levels of both HRG receptors. Although Hst blocked HRG signaling in both parental and HER-2 transfected cells, it enhanced sensitivity to tamoxifen only in the MCF7 cells that overexpressed HER-2. To evaluate further the efficacy of Hst as an anticancer agent, His-tagged Hst was expressed in transfected insect cells, purified, and added to the breast cancer cells. As in the transfected cells, purified Hst inhibited HER-3 levels and suppressed HRG-induced proliferation of MCF7 and BT474 breast cancer cells. In contrast, the HER-2 monoclonal antibody, herceptin, downregulated HER-2, but not HER-3. These results suggest the potential use of Hst against HRG-mediated growth of breast cancers with high and low levels of HER-2 and against tamoxifen resistance in HER-2 overexpressing breast cancer.

Requirement for ErbB2/ErbB signaling in developing cartilage and bone

During endochondral ossification, the skeletal elements of vertebrate limbs form and elongate via coordinated control of chondrocyte and osteoblast differentiation and proliferation. The role of signaling by the ErbB family of receptor tyrosine kinases, which consists of ErbB1 (epidermal growth factor receptor or EGFR), ErbB2, ErbB3 and ErbB4, has been little studied during cartilage and bone development. Signaling by the ErbB network generates a diverse array of cellular responses via formation of ErbB dimers activated by distinct ligands that produce distinct signal outputs. Herstatin is a soluble ErbB2 receptor that acts in a dominant negative fashion to inhibit ErbB signaling by binding to endogenous ErbB receptors, preventing functional dimer formation. Here, we examine the effects of Herstatin on limb skeletal element development in transgenic mice, achieved via Prx1 promoter‐driven expression in limb cartilage and bone. The limb skeletal elements of Prx1‐Herstatin embryos are shortened, and chondrocyte maturation and osteoblast differentiation are delayed. In addition, proliferation by chondrocytes and periosteal cells of Prx1‐Herstatin limb skeletal elements is markedly reduced. Our study identifies requirements for ErbB signaling in the maintenance of chondrocyte and osteoblast proliferation involved in the timely progression of chondrocyte maturation and periosteal osteoblast differentiation.

Insulin and epidermal growth factor stimulate phosphorylation of p185HER-2 in the breast carcinoma cell line, BT474

The product of the HER-2 proto-oncogene, p185HER-2, was found to be amplified approximately 10-fold in the human breast carcinoma cell line, BT474, compared to a cell line, HBL-100, derived from normal breast tissue. To explore the possible role of p185HER-2 in growth of the breast carcinoma cells, we investigated factors that may modulate cell growth and phosphorylation of the HER-2 protein product. Two growth factors, epidermal growth factor (EGF) and insulin, stimulated phosphorylation of the HER-2 protein product. In response to insulin, the phosphoserine and phosphothreonine content in p185HER-2 was transiently enhanced about 6-fold. When EGF was added to BT474 cells there was 2- to 3-fold enhanced phosphorylation of serine and threonine residues in p185HER-2 which was maintained for at least 60 min. Although p185HER-2 has been found to be phosphorylated on tyrosine residues following EGF treatment of several different cell types, we estimate that less than 1% of the protein contained phosphotyrosine in the BT474 cells.

Receptor binding specificities of Herstatin and its intron 8-encoded domain

Retention of intron 8 in alternative HER‐2 mRNA generates an inhibitory secreted ligand, Herstatin, with a novel receptor‐binding domain (RBD) encoded by the intron. This study examines binding interactions with several receptors and investigates sequence variations in intron 8. The RBD, expressed as a peptide, binds at nM concentrations to HER‐2, the EGFR, ΔEGFR, HER‐4 and to the IGF‐1 receptor, but not to HER‐3 nor to the FGF‐3 receptor, whereas a rare mutation in the RBD (Arg to Ile) eliminates receptor binding. The full‐length Herstatin binds with 3–4‐fold higher affinity than its RBD, but with ∼10‐fold lower affinity to the IGF‐IR. Sequence conservation in rhesus monkey but not in rat suggests that intron 8 recently diverged as a receptor‐binding module critical for the function of Herstatin.