Peptide Ligands Specifically Targeting HER2 Receptor and the Role Played by a Synthetic Model System of the Receptor Extracellular Domain: Hypothesized Future Perspectives.

A short (Fab)trastuzumab-derived peptide specific for HER2 receptor was identified. Its affinity for the model system HER2-DIVMP was found in a nanomolar range. The structural determinants responsible for the interaction between this ligand (A9) and HER2-DIVMP were investigated by both computational and NMR analysis. Next, the possibility of using A9 as HER2- specific probe for the nuclear medicine imaging was evaluated by conjugating A9 with the DTPA chelator and radiolabeling it with 111In. The developed probe retained a nanomolar affinity to HER2-overexpressing cancer cells, however, some unspecific binding also occurred. The peptide internalization into cells by receptor-mediated endocytosis was also studied. Future perspectives are aimed at using A9 as a probe for molecular imaging diagnostics as well as active targeting of anticancer drugs. Lead structure optimization is needed to minimize the percentage of A9 unspecific binding and to increase the binding affinity to the receptor.

HER2 targeted biological macromolecule modified liposomes for improved efficacy of capecitabine in breast cancer.

The present research reports the beneficial effects of surface modified chitosan and tumor-homing peptide conjugated liposomes of capecitabine (CAP) for treating breast cancer. Liposomal formulation of CAP was prepared by film hydration method using cholesterol-THP conjugate (CTHP-CAP-LPs) to achieve active targeting through HER2 receptors. CTHP-CAP-LPs significantly improved the specificity and efficacy of CAP by improving cell uptake, cytotoxicity and tumor regression in tumor bearing mice. CTHP-CAP-LPs, therefore, is a promising approach to improve the anticancer effects of CAP.

Reducing the Detrimental Effects of Saturation Phenomena in FRET Microscopy.

Although Förster resonance energy transfer (FRET) is one of the most widely used biophysical methods in biology, the effect of high excitation intensity, leading to donor and acceptor saturation, has not been addressed previously. Here, we present a formalism for the experimental determination of the FRET efficiency at high excitation intensity when saturation of both the donor and the acceptor significantly affect conventional FRET calculations. We show that the proposed methodology significantly reduces the dependence of the FRET efficiency on excitation intensity, which otherwise significantly distorts FRET calculations at high excitation intensities commonly used in experiments. The work presented here adds additional rigor to the FRET-based investigation of protein interactions and strengthens the device independence of such results.

Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin.

OBJECTIVE: Long-term use of doxorubicin (Dox) can cause neurobiological side effects associated with depression, but the underlying mechanisms remain equivocal. While recent evidence has indicated that Neuregulin-1 (NRG1) and its ErbB receptors play an essential role in neural function, much is still unknown concerning the biological link between the NRG1/ErbB pathway and the Dox-induced neurotoxicity. Therefore, we examined the protein expression of NRG1 and ErbB receptors in the hippocampus of rats following Dox treatment. MATERIALS AND METHODS: The drug was administered every 2 days at a dose of 2.5 mg/kg, and the animals in different groups were treated with intraperitoneal injection for three or seven times, respectively. RESULTS: Our data showed that the rats treated with Dox for seven times (DoxL group) exhibited depression-like behaviors, whereas the short-term treatment (DoxS group) had no effect on the behavioral changes. Dox treatment also induced the neural apoptosis with more severe neurotoxicity. Intriguingly, the expression of NRG1 and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 were significantly decreased in the DoxL group, but enhanced activation of ErbB receptors was observed in the DoxS group. In parallel, administration of Dox for seven times suppressed the downstream Akt and ERK phosphorylation, while the Akt phosphorylation was enhanced with the administration of Dox for three times. CONCLUSION: Our data first showed the Dox-induced alterations of the NRG1/ErbB system in the hippocampus, indicating the potential involvement of the NRG1/ErbB pathway in the Dox-induced nervous system dysfunction.

Post-transcriptional regulation of ERBB2 by miR26a/b and HuR confers resistance to tamoxifen in estrogen receptor-positive breast cancer cells.

Tamoxifen-resistant (TAMR) estrogen receptor-positive (ER+) breast cancer is characterized by elevated Erb-B2 receptor tyrosine kinase 2 (ERBB2) expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the TAMR cells remain poorly understood. Herein, we reported that the ERBB2 expression is regulated at the post-transcriptional level by miR26a/b and the RNA-binding protein human antigen R (HuR), both of which associate with the 3'-UTR of the ERBB2 transcripts. We demonstrated that miR26a/b inhibits the translation of ERBB2 mRNA, whereas HuR enhances the stability of the ERBB2 mRNA. In TAMR ER+ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER+ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER+ breast cancer cells is attributable to an increase in the HuR mRNA isoform with shortened 3'-UTR, which exhibits increased translational activity. This shortening of the HuR mRNA 3'-UTR via alternative polyadenylation (APA) was observed to be dependent on cleavage stimulation factor subunit 2 (CSTF2/CstF-64), which is up-regulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR post-transcriptionally up-regulates ERBB2 expression in TAMR ER+ breast cancer cells.

Interplay of the iron-regulated metastasis suppressor NDRG1 with epidermal growth factor receptor (EGFR) and oncogenic signaling.

The iron-regulated metastasis suppressor N-myc downstream-regulated gene 1 (NDRG1) has been shown to inhibit numerous oncogenic signaling pathways in cancer cells. Recent findings have demonstrated that NDRG1 inhibits the ErbB family of receptors, which function as key inducers of carcinogenesis. NDRG1 attenuates ErbB signaling by inhibiting formation of epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) and HER2/HER3 heterodimers and by down-regulating EGFR via a mechanism involving its degradation. Understanding the complex interplay between NDRG1, iron, and ErbB signaling is vital for identifying novel, more effective targets for cancer therapy.

A functional signal profiling test for identifying a subset of HER2-negative breast cancers with abnormally amplified HER2 signaling activity.

The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2-, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2- breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%-37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways.

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-ß pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.

[Cancer gene therapy with plasmid expressing anti-HER2/neu-toxin].

The HER2/neu receptor is over-expressed on the surface of many types of cancer cells, and is widely used for tar- geted delivery of anticancer drugs. We have created geneti- cally engineered construct expressing the PE40 fragment of Pseudomonas toxin bound with the DARPin molecule which recognizes the HER2/neu receptor with high specificity. The construct destroyed transfected tumor cells in vitro. Intra-tumor injections of the construct complexed with polyethyleneimine led to growth retardation of D2F2/E2 tumors in mice. These results suggest a possibility of using this approach to develop new anticancer drugs.

ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation.

The murine neonatal heart can regenerate after injury through cardiomyocyte (CM) proliferation, although this capacity markedly diminishes after the first week of life. Neuregulin-1 (NRG1) administration has been proposed as a strategy to promote cardiac regeneration. Here, using loss- and gain-of-function genetic tools, we explore the role of the NRG1 co-receptor ERBB2 in cardiac regeneration. NRG1-induced CM proliferation diminished one week after birth owing to a reduction in ERBB2 expression. CM-specific Erbb2 knockout revealed that ERBB2 is required for CM proliferation at embryonic/neonatal stages. Induction of a constitutively active ERBB2 (caERBB2) in neonatal, juvenile and adult CMs resulted in cardiomegaly, characterized by extensive CM hypertrophy, dedifferentiation and proliferation, differentially mediated by ERK, AKT and GSK3ß/ß-catenin signalling pathways. Transient induction of caERBB2 following myocardial infarction triggered CM dedifferentiation and proliferation followed by redifferentiation and regeneration. Thus, ERBB2 is both necessary for CM proliferation and sufficient to reactivate postnatal CM proliferative and regenerative potentials.

Transactivation of ErbB receptors by leptin in the cardiovascular system: mechanisms, consequences and target for therapy.

Many experimental and clinical studies have demonstrated that elevated leptin concentration in patients with obesity/metabolic syndrome contributes to the pathogenesis of cardiovascular disorders including arterial hypertension, atherosclerosis, restenosis after coronary angioplasty and myocardial hypertrophy. Receptor tyrosine kinases belonging to the ErbB family, especially ErbB1 (epidermal growth factor receptor) and ErbB2 are abundantly expressed in the blood vessels and the heart. EGFR is activated not only by its multiple peptide ligands but also by many other factors including angiotensin II, endothelin-1, norepinephrine, thrombin and prorenin; the phenomenon referred to as "transactivation". Augmented EGFR signaling contributes to abnormalities of vascular tone and renal sodium handling as well as vascular remodeling and myocardial hypertrophy through various intracellular mechanisms, in particular extracellular signal-regulated kinases (ERK) and phosphoinositide 3-kinase (PI3K). Recent experimental studies indicate that chronically elevated leptin transactivates the EGFR through the mechanisms requiring reactive oxygen species and cytosolic tyrosine kinase, c-Src. In addition, hyperleptinemia increases ErbB2 activity in the arterial wall. Stimulation of EGFR and ErbB2 downstream signaling pathways such as ERK and PI3K in the vascular wall and the kidney may contribute to the increase in vascular tone, enhanced tubular sodium reabsorption as well as vascular and renal lesions in hyperleptinemic obese subjects.

Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation.

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

Cdk2 and Pin1 negatively regulate the transcriptional corepressor SMRT.

Silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) is a transcriptional corepressor that participates in diverse signaling pathways and human diseases. However, regulation of SMRT stability remains largely unexplored. We show that the peptidyl-prolyl isomerase Pin1 interacts with SMRT both in vitro and in mammalian cells. This interaction requires the WW domain of Pin1 and SMRT phosphorylation. Pin1 regulates SMRT protein stability, thereby affecting SMRT-dependent transcriptional repression. SMRT phosphorylation at multiple sites is required for Pin1 interaction, and these sites can be phosphorylated by Cdk2, which interacts with SMRT. Cdk2-mediated phosphorylation of SMRT is required for Pin1 binding and decreases SMRT stability, whereas mutation of these phosphorylation sites abrogates Pin1 binding and stabilizes SMRT. Finally, decreases in SMRT stability occur in response to the activation of Her2/Neu/ErbB2, and this receptor functions upstream of both Pin1 and Cdk2 in the signaling cascade that regulates SMRT stability and cellular response to tamoxifen.

Quantifying the effects of co-expressing EGFR and HER2 on HER activation and trafficking.

The human epidermal growth factor receptor (HER) system is an intricately regulated system that plays critical roles in development and tumorigenesis. Here, we apply integrated experimentation and modeling to analyze HER receptor activation in a panel of cell lines expressing endogenous levels of EGFR/HER1 and different levels of HER2. A mathematical model that includes the fundamental processes involved in receptor activation and trafficking was used to fit the experimental data, and values of the independent parameters for active receptor dimer formation affinities, trafficking rates and relative phosphorylation levels were estimated. Obtained parameter values quantitatively support the existing ideas on the effect of HER2 on EGFR dynamics, and enable us to predict the abundances of various phosphorylated receptor dimers in the cell lines.

Neuregulin-1 enhances survival of human astrocytic glioma cells.

Malignant astrocytic gliomas, referred to as astrocytomas, represent the most commonly diagnosed adult primary brain tumor. These tumors are characterized by unrelenting growth that is often resistant to chemotherapy and radiation therapy. Tumor expansion into the healthy surrounding brain tissue produces severe and often fatal consequences. In this study, we examine the potential for the neuregulin-1/erbB receptor signaling cascade to contribute to this process by modulating glioma cell growth. Using antibodies specific for the erbB receptors, we demonstrate the expression patterns for the erbB2, erbB3, and erbB4 receptors in human glioma biopsy samples. We then verify receptor expression in a panel of human glioma cell lines. Next, we investigate the status of the erbB2 and erbB3 receptors in the human glioma cell lines and find that they are constitutively tyrosine-phosphorylated and heterodimerized. Subsequently, we demonstrate that theses same cell lines express membrane bound and released forms of neuregulins, the erbB receptor ligands, suggesting a possible autocrine or paracrine signaling network. Furthermore, we show that exogenous activation of erbB2 and erbB3 receptors in U251 glioma cells by recombinant Nrg-1beta results in enhanced glioma cell growth under conditions of serum-deprivation. This enhancement is due to an increase in cell survival rather than an increase in cell proliferation and is dependent on the activation of erbB2 and phosphatidylinositol-3 kinase (PI3K). Moreover, Nrg-1beta activates an inhibitor of apoptosis, Akt, implying a possible role for this kinase in mediating Nrg-1beta effects in gliomas. This data suggests that glioma cells may use autocrine or paracrine neuregulin-1/erbB receptor signaling to enhance cell survival under conditions where growth would otherwise be limited.

Activation of the phosphatidylinositol 3-kinase/Akt pathway prevents radiation-induced apoptosis in breast cancer cells.

Radiotherapy is widely used in the treatment of breast cancer and reduces the risk of loco-regional recurrence. Overexpression of the erbB2 receptor occurs in 20-30% of all breast cancers, and seems to be involved in chemotherapeutic resistance of breast cancer cells and radioresistance of lung cancer cells. The hypothesis of this study was that erbB2 confers resistance to radiation-induced apoptosis in breast cancer cells through the phosphatidylinositol 3-kinase (PI3-K)/Akt signalling pathway. Two human breast cancer cell lines were used, BT-474 and MCF-7. BT-474 cells overexpress erbB2 and have mutated p53, while MCF-7 have normal expression of erbB2 and functional p53. The cells were treated with the PI3-K inhibitor wortmannin or the erbB receptor ligand heregulin-beta1, which is expressed by both malignant and stromal cells in vivo. After pharmacological treatment, the cells were irradiated with 10 Gy gamma-radiation. Consistent with the p53 status in the cell lines, gamma-radiation caused G1 arrest in MCF-7 cells, but not in BT-474 cells. 10 Gy gamma-radiation increased apoptosis by on an average 76% (95% CI, 44-109%) in MCF-7. Treatment of MCF-7 with heregulin-beta1 decreased apoptosis by 66% (95% CI, 48-84%) compared to the untreated controls. In BT-474 cells, wortmannin in combination with radiation resulted in 119% (95% CI, 76-161%) more apoptosis compared to wortmannin alone, whereas radiation alone resulted in 45% (95% CI, 15-75%) increased apoptosis. This radiosensitising effect was not seen in MCF-7. Furthermore, transfection of MCF-7 cells with constitutively active Akt made the cells more resistant against apoptosis. Taken together, our results support the hypothesis that the erbB2/PI3-K/Akt signalling pathway is involved in resistance to radiation-induced apoptosis in breast cancer cells in which this signalling pathway is overstimulated.

Axonal neuregulin signals cells of the oligodendrocyte lineage through activation of HER4 and Schwann cells through HER2 and HER3.

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor-inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite-type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.