PANTHER: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a therapeutic target to which HER2/HER3 activation may contribute resistance. This Phase I/II study examined the toxicity and efficacy of high-dose pulsed AZD8931, an EGFR/HER2/HER3 inhibitor, combined with chemotherapy, in metastatic colorectal cancer (CRC). METHODS: Treatment-naive patients received 4-day pulses of AZD8931 with irinotecan/5-FU (FOLFIRI) in a Phase I/II single-arm trial. Primary endpoint for Phase I was dose limiting toxicity (DLT); for Phase II best overall response. Samples were analysed for pharmacokinetics, EGFR dimers in circulating exosomes and Comet assay quantitating DNA damage. RESULTS: Eighteen patients received FOLFIRI and AZD8931. At 160 mg bd, 1 patient experienced G3 DLT; 160 mg bd was used for cohort expansion. No grade 5 adverse events (AE) reported. Seven (39%) and 1 (6%) patients experienced grade 3 and grade 4 AEs, respectively. Of 12 patients receiving 160 mg bd, best overall response rate was 25%, median PFS and OS were 8.7 and 21.2 months, respectively. A reduction in circulating HER2/3 dimer in the two responding patients after 12 weeks treatment was observed. CONCLUSIONS: The combination of pulsed high-dose AZD8931 with FOLFIRI has acceptable toxicity. Further studies of TKI sequencing may establish a role for pulsed use of such agents rather than continuous exposure. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT01862003.

A novel anti-HER2 antibody GB235 reverses Trastuzumab resistance in HER2-expressing tumor cells in vitro and in vivo.

HER2 overexpression is frequently associated with tumor metastasis and poor prognosis of breast cancer. More evidence indicates that HER3 is involved in HER2-resistant therapies. Combination treatments with two or more different monoclonal antibodies are a promising strategy to overcome resistance to HER2 therapies. We presented a novel fully human HER2-targeted monoclonal antibody, GB235, screened from a phage-display library against the HER2 antigen. GB235 in combination with Trastuzumab overcomes resistance in HER2-positive tumors and results in more sustained inhibition of tumor growth over time. The competition binding assay showed that the epitopes of GB235 do not overlap with those of Pertuzumab and Trastuzumab on HER2. Further HER2 mutagenesis results revealed that the binding epitopes of GB235 were located in the domain III of HER2. The mechanism of action of GB235 in blocking HER2-driven tumors is different from the mechanisms of Trastuzumab or Pertuzumab. GB235 does not affect the heterodimerization of HER2 and HER3, whereas the GB235 combined treatment with Trastuzumab significantly inhibited heregulin-induced HER3 phosphorylation and downstream signaling. Moreover, GB235 in combination with Trastuzumab reversed the resistance to heregulin-induced proliferation in HER2-overexpressing cancer cell lines. GB235 combined with Trastuzumab treatment in xenograft models resulted in improved antitumor activity. Complete tumor suppression was observed in the HER2-positive NCI-N87 xenograft model treated with the combination treatment with GB235 and Trastuzumab. In a Trastuzumab-resistant patient-derived tumor xenograft model GA0060, GB235 plus Trastuzumab reversed the resistance to Trastuzumab monotherapy. Because GB235 showed a different working mechanism with Pertuzumab and Trastuzumab, these agents can be considered complementary therapy against HER2 overexpression tumors.

Synergistic anticancer effect of combined use of Trichosanthes kirilowii with cisplatin and pemetrexed enhances apoptosis of H1299 non-small-cell lung cancer cells via modulation of ErbB3.

BACKGROUND: Lung cancer is one of the most common malignancies worldwide. To treat lung cancer, various anticancer drugs were developed and tested, but they failed because of drug resistance. In the present study, we tested herbal medicines, such as TK and CuD, as anticancer drugs to decrease side effects and resistance. METHODS: Cell viability was measured by an MTT assay. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by an annexin V-FITC/PI assay. We performed RTK kit analysis. Levels of p-ErbB3, p-STAT3, p-NF-κB, and caspases were measured by western blot analysis. Nuclear staining of ErbB3 was measured by immunocytochemistry. Transcriptional activity of STAT3 and NF-κB was detected by STAT3 and NF-κB luciferase reporter gene assays. RESULTS: We found a synergistic effect of TK with CDDP and PXD in primary culture of human NSCLC tumor cells. The combination of CDDP/PXD and TK or CuD inhibited the proliferation of H1299 cells. The combination of CDDP/PXD and TK or CuD induced sub-G1 and G2/M cell cycle arrest in H1299 cells. The combination of CDDP/PXD and TK or CuD induced apoptosis, regulated apoptotic molecules, caused morphological changes and inhibited colony formation in H1299 cells. We found that TK suppresses p-ErbB3 expression and signaling. The combination of CDDP/PXD and TK or CuD inhibited p-AKT, p-Erk, and p-JNK signaling and suppressed Stat3 and NF-κB transcriptional activity in H1299 cells. More importantly, the combination of CDDP/PXD and TK or CuD inhibited p-ErbB3 and downstream molecules in H1299 cells. The combination of CDDP/PXD and TK or CuD inhibited ErbB2/ErbB3 dimerization. Our results clearly demonstrate that the synergistic effect of CDDP/PXD and TK or CuD inhibits cell growth and induces apoptosis by inhibiting ErbB3 signaling. CONCLUSION: The combination of CDDP/PXD and TK or CuD decreases cell proliferation and induces apoptosis by inhibiting ErbB3 signaling in H1299 lung cancer cells. TK or CuD could be useful as a compound to treat lung cancer. Additionally, targeting ErbB3 may also be useful for treating lung cancer.

TAS0728, A Covalent-binding, HER2-selective Kinase Inhibitor Shows Potent Antitumor Activity in Preclinical Models.

Activated HER2 is a promising therapeutic target for various cancers. Although several reports have described HER2 inhibitors in development, no covalent-binding inhibitor selective for HER2 has been reported. Here, we report a novel compound TAS0728 that covalently binds to HER2 at C805 and selectively inhibits its kinase activity. Once TAS0728 bound to HER2 kinase, the inhibitory activity was not affected by a high ATP concentration. A kinome-wide biochemical panel and cellular assays established that TAS0728 possesses high specificity for HER2 over wild-type EGFR. Cellular pharmacodynamics assays using MCF10A cells engineered to express various mutated HER2 genes revealed that TAS0728 potently inhibited the phosphorylation of mutated HER2 and wild-type HER2. Furthermore, TAS0728 exhibited robust and sustained inhibition of the phosphorylation of HER2, HER3, and downstream effectors, thereby inducing apoptosis of HER2-amplified breast cancer cells and in tumor tissues of a xenograft model. TAS0728 induced tumor regression in mouse xenograft models bearing HER2 signal-dependent tumors and exhibited a survival benefit without any evident toxicity in a peritoneal dissemination mouse model bearing HER2-driven cancer cells. Taken together, our results demonstrated that TAS0728 may offer a promising therapeutic option with improved efficacy as compared with current HER2 inhibitors for HER2-activated cancers. Assessment of TAS0728 in ongoing clinical trials is awaited (NCT03410927).

Potential anti-proliferative activity of AgNPs synthesized using M. longifolia in 4T1 cell line through ROS generation and cell membrane damage.

To overcome the problem of breast cancer, silver nanoparticles (AgNPs) synthesized using Indian medicinal plant Madhuca longifolia could be explored as an alternative anticancer medicine. Synthesized AgNPs were studied their characteristics and their anti-proliferative property was investigated in breast cancer cell line (4T1). Based on zeta sizer analysis, the size of the AgNPs was 103.5 nm and potential -9.57 eV. Fe-SEM results showed particle size of 69.4-99.4 nm while TEM images indicated the particle size of 18-24 nm. In dose-dependent study, AgNPs showed 93% of anti-proliferative activity at 50 µg/ml whereas the methanolic extract of M. longifolia showed 80% activity only at 10-fold increased concentration (500 µg/ml). AgNPs exhibited higher level of cytotoxicity in breast cancer cell line than extract through cell wall degradation and ROS generation. Such effective AgNPs could be investigated further through in vivo models with a view to develop anticancer drug.

Dual Inhibition of IGF-1R and ErbB3 Enhances the Activity of Gemcitabine and Nab-Paclitaxel in Preclinical Models of Pancreatic Cancer.

Purpose: Insulin-like growth factor receptor 1 (IGF-1R) is critically involved in pancreatic cancer pathophysiology, promoting cancer cell survival and therapeutic resistance. Assessment of IGF-1R inhibitors in combination with standard-of-care chemotherapy, however, failed to demonstrate significant clinical benefit. The aim of this work is to unravel mechanisms of resistance to IGF-1R inhibition in pancreatic cancer and develop novel strategies to improve the activity of standard-of-care therapies.Experimental Design: Growth factor screening in pancreatic cancer cell lines was performed to identify activators of prosurvival PI3K/AKT signaling. The prevalence of activating growth factors and their receptors was assessed in pancreatic cancer patient samples. Effects of a bispecific IGF-1R and ErbB3 targeting antibody on receptor expression, signaling, cancer cell viability and apoptosis, spheroid growth, and in vivo chemotherapy activity in pancreatic cancer xenograft models were determined.Results: Growth factor screening in pancreatic cancer cells revealed insulin-like growth factor 1 (IGF-1) and heregulin (HRG) as the most potent AKT activators. Both growth factors reduced pancreatic cancer cell sensitivity to gemcitabine or paclitaxel in spheroid growth assays. Istiratumab (MM-141), a novel bispecific antibody that blocks IGF-1R and ErbB3, restored the activity of paclitaxel and gemcitabine in the presence of IGF-1 and HRG in vitro Dual IGF-1R/ErbB3 blocking enhanced chemosensitivity through inhibition of AKT phosphorylation and promotion of IGF-1R and ErbB3 degradation. Addition of istiratumab to gemcitabine and nab-paclitaxel improved chemotherapy activity in vivoConclusions: Our findings suggest a critical role for the HRG/ErbB3 axis and support the clinical exploration of dual IGF-1R/ErbB3 blocking in pancreatic cancer. Clin Cancer Res; 24(12); 2873-85. ©2018 AACR.

Non invasive imaging assessment of the biodistribution of GSK2849330, an ADCC and CDC optimized anti HER3 mAb, and its role in tumor macrophage recruitment in human tumor-bearing mice.

The purpose of this work was to use various molecular imaging techniques to non-invasively assess GSK2849330 (anti HER3 ADCC and CDC enhanced 'AccretaMab' monoclonal antibody) pharmacokinetics and pharmacodynamics in human xenograft tumor-bearing mice. Immuno-PET biodistribution imaging of radiolabeled 89Zr-GSK2849330 was assessed in mice with HER3 negative (MIA-PaCa-2) and positive (CHL-1) human xenograft tumors. Dose dependency of GSK2849330 disposition was assessed using varying doses of unlabeled GSK2849330 co-injected with 89Zr-GSK2849330. In-vivo NIRF optical imaging and ex-vivo confocal microscopy were used to assess the biodistribution of GSK2849330 and the HER3 receptor occupancy in HER3 positive xenograft tumors (BxPC3, and CHL-1). Ferumoxytol (USPIO) contrast-enhanced MRI was used to investigate the effects of GSK2849330 on tumor macrophage content in CHL-1 xenograft bearing mice. Immuno-PET imaging was used to monitor the whole body drug biodistribution and CHL-1 xenograft tumor uptake up to 144 hours post injection of 89Zr-GSK2849330. Both hepatic and tumor uptake were dose dependent and saturable. The optical imaging data in the BxPC3 xenograft tumor confirmed the tumor dose response finding in the Immuno-PET study. Confocal microscopy showed a distinguished cytoplasmic punctate staining pattern within individual CHL-1 cells. GSK2849330 inhibited tumor growth and this was associated with a significant decrease in MRI signal to noise ratio after USPIO injection and with a significant increase in tumor macrophages as confirmed by a quantitative immunohistochemistry analysis. By providing both dose response and time course data from both 89Zr and fluorescently labeled GSK2849330, complementary imaging studies were used to characterize GSK2849330 biodistribution and tumor uptake in vivo. Ferumoxytol-enhanced MRI was used to monitor aspects of the immune system response to GSK2849330. Together these approaches potentially provide clinically translatable, non-invasive techniques to support dose optimization, and assess immune activation and anti-tumor responses.

Discovery of ERBB3 inhibitors for non-small cell lung cancer (NSCLC) via virtual screening.

As a member of the epidermal growth factor receptor family (EGFR) of receptor tyrosine kinases, ERBB3 plays an important role in mediating cellular growth and differentiation. Recent research works identified that CD74-NRG1 fusions lead to overexpression of the EGF-like domain of NRG1, and thus activate ERBB3 and PI3K-AKT signaling pathways. The fusion was detected in lung adenocarcinomas, and served as an important oncogenic factor for ERBB3 driven cancers. A sequential virtual screening strategy has been applied to ERBB3 crystal structure using databases of natural products and Chinese traditional medicine compounds, and led to identification of a group of small molecular compounds potentially capable of blocking ERBB3. Six small molecular compounds were selected for in vitro analysis. Five of these molecules significantly inhibited the growth of A549 cells. Among them, compound VS1 is the most promising one with IC50 values of 269.75 µM, comparing to the positive control of nimustine hydrochloride with IC50 values of 264.14 µM. With good specificity and predicted ADMET results, our results support the feasibility by using a pharmacophore of the compound VS1 for designing and optimization of ERBB3 inhibitors.

Neuregulin 1 confers neuroprotection in SOD1-linked amyotrophic lateral sclerosis mice via restoration of C-boutons of spinal motor neurons.

INTRODUCTION: Increasing evidence implicates the role of the cell types surrounding motor neurons, such as interneurons and glial cells, in non-cell autonomous neurodegeneration of amyotrophic lateral sclerosis (ALS). C-boutons, the large cholinergic synapses that innervate spinal α-motor neurons to control their excitability, are progressively lost from motor neurons in both human ALS and mutant Cu/Zn superoxide dismutase 1 (SOD1)-ALS mice. Neuregulin-1 (NRG1), a trophic factor implicated in neural development, transmission, and synaptic plasticity, has been reported to localize in the synapse of C-boutons. However, the roles of NRG1 in maintenance of motor neuron health and activity, as well as the functional consequences of its alteration in motor neuron disease, are not fully understood. RESULTS: NRG1 was localized to the post-synaptic face of C-boutons and its expression was significantly lost in SOD1-ALS mice and human ALS patients. Losses of NRG1 expression and C-boutons occurred almost contemporaneously in SOD1-ALS mice. In addition, expressions of ErbB3 and ErbB4, receptors for NRG1, were reduced in the motor neurons of SOD1-ALS mice. Furthermore, viral-mediated delivery of type III-NRG1 to the spinal cord restored the number of C-boutons and extended the survival time of SOD1-ALS mice. CONCLUSIONS: These results suggest that maintenance of NRG1-ErbB4/3 axis by supplementation of NRG1 confers neuroprotection in motor neuron disease, partly through the maintenance of C-boutons of spinal motor neurons.

Inhibition of the HER2 pathway by n-3 polyunsaturated fatty acids prevents breast cancer in fat-1 transgenic mice.

Overexpression of the tyrosine kinase receptor, ErbB2/HER2/Neu, occurs in 25-30% of invasive breast cancer (BC) with poor patient prognosis. Due to confounding factors, inconsistencies still remain regarding the protective effects of n-3 polyunsaturated fatty acids (PUFAs) on BC. We therefore evaluated whether fat-1 transgenic mice, endogenously synthesizing n-3 PUFAs from n-6 PUFAs, were protected against BC development, and we then aimed to study in vivo a mechanism potentially involved in such protection. E0771 BC cells were implanted into fat-1 and wild-type (WT) mice. After tumorigenesis examination, we analyzed the expression of proteins involved in the HER2 signaling pathway and lipidomic analyses were performed in tumor tissues and plasma. Our results showed that tumors totally disappeared by day 15 in fat-1 mice but continued to grow in WT mice. This prevention can be related in part to significant repression of the HER2/ß-catenin signaling pathway and formation of significant levels of n-3 PUFA-derived bioactive mediators (particularly 15-hydroxyeicosapentaenoic acid, 17-hydroxydocosahexaenoic acid, and prostaglandin E3) in the tumors of fat-1 mice compared with WT mice. All together these data demonstrate an anti-BC effect of n-3 PUFAs through, at least in part, HER2 signaling pathway downregulation, and highlight the importance of gene-diet interactions in BC.

ErbB receptors and PKC regulate PC12 neuronal-like differentiation and sodium current elicitation.

Excitability, neurite outgrowth and their specification are very important features in the establishment of neuronal differentiation. We have studied a conditioned medium (CM) from sciatic nerve which is able to induce a neuronal-like differentiation of PC12 cells. Previously, we have demonstrated that supplementing this CM with a generic inhibitor (k252a), which mainly inhibits tropomyosin-related kinase receptors (Trk receptors) and protein kinase C (PKC), caused neurite elongation, sodium current induction and axon development. In the present work, we are showing that the enhancement of neurite length and induction of sodium currents induced by CM+k252a were prevented by ErbB receptor inhibition. Additionally, we demonstrated that specific inhibition of PKC produced a similar effect to that exerted by k252a in CM-treated cells, specifically by increasing the percentage of differentiated cells with long neurites and inducing sodium currents. Moreover, CM changed the mRNA levels for ErbB2 and ErbB3 increasing them 6- and 36-folds respectively compared to their control. The inclusion of k252a with CM changed the ErbB1, ErbB2 and ErbB3 mRNA proportions increasing those eight-, seven- and fivefolds respectively. From this point, it is clear that appropriate ErbB receptor levels and PKC inhibition are necessary to enhance the effect of the CM in inducing the neuronal-like differentiation of PC12 cells. In summary, we demonstrated the involvement of ErbB receptors in the regulation of neurite elongation and sodium current induction in PC12 cells and propose that these processes could be initiated by ErbB receptors followed by a fine regulation of PKC signaling. These findings might implicate a novel interplay between ErbB receptors and PKC in the regulation of these molecular mechanisms.

Apple procyanidins affect several members of the ErbB receptor tyrosine kinase family in vitro.

Complex polyphenol-rich extracts from apples are known to inhibit the activity of the epidermal growth factor receptor (EGFR) in vitro. The aim of the present study was to identify the bioactive constituents of the apple juice extract which contribute substantially to this potentially chemopreventive effect and to address the question whether the effect is specific to the EGFR or whether other members of the ErbB-receptor family might also be affected. Apple-derived dihydrochalcones and their respective glycosides were found to decrease EGFR activity under cell-free conditions with IC50-values ranging from 0.4 ± 0.1 to 267.0 ± 50.0 µM but showed no activity on human cancer cells. The concentration of quercetin or its glycosides in the extract was too low to contribute substantially to the EGFR-inhibitory properties. In contrast, fractions derived from the apple juice extract comprising ≥86% oligomeric procyanidins (OPCs) suppressed the activity of the EGFR in cell culture with an IC50 ∼ 100 µg mL(-1). In addition, the activity of further members of the ErbB-receptor family was potently inhibited, with ErbB3 receptor activity being most potently decreased (IC50 ∼ 10 µg mL(-1)). From the apple polyphenols identified so far OPCs were found to add the highest contribution to the inhibitory effects towards members of the ErbB-receptor family. Considering the crucial role of the ErbB-receptors in carcinogenesis, these results support the hypothesis that apple-derived OPCs as well as OPC-rich apple preparations might be of interest with respect to chemoprevention.

Study on predictive role of AR and EGFR family genes with response to neoadjuvant chemotherapy in locally advanced breast cancer in Indian women.

Locally advanced breast cancer (LABC) remains a clinical challenge as the majority of patients with this diagnosis develop distant metastases despite appropriate therapy. We analyzed expression of steroid and growth hormone receptor genes as well as gene associated with metabolism of chemotherapeutic drugs in locally advanced breast cancer before and after neoadjuvant chemotherapy (NACT) to study whether there is a change in gene expression induced by chemotherapy and whether such changes are associated with tumor response or non-response. Fifty patients were included with locally advanced breast cancer treated with cyclophosphamide, adriamycin, 5-fluorouracil (CAF)-based neoadjuvant chemotherapy before surgery. Total RNA was extracted from 50 match samples of pre- and post-NACT tumor tissues. RNA expression levels of epidermal growth factor receptor family genes including EGFR, ERBB2, ERBB3, androgen receptor (AR), and multidrug-resistance gene 1 (MDR1) were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. Responders show significantly high levels of pre-NACT AR gene expression (P = 0.016), which reduces following NACT (P = 0.008), and hence can serve as a useful tool for the prediction of the success of neoadjuvant chemotherapy in individual cancer patients with locally advanced breast carcinoma. Moreover, a significant post-therapeutic increase in the expression levels of EGFR and MDR1 gene in responders (P = 0.026 and P < 0.001) as well as in non-responders (P = 0.055, P = 0.001) suggests that expression of these genes changes during therapy but they do not have any impact on tumor response, whereas a post-therapeutic reduction was observed in AR in responders. This indicates an independent predictive role of AR with response to NACT.

During hormone depletion or tamoxifen treatment of breast cancer cells the estrogen receptor apoprotein supports cell cycling through the retinoic acid receptor α1 apoprotein.

INTRODUCTION: Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear. METHODS: In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR. RESULTS: Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-ß and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions. CONCLUSIONS: In hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RARα1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RARα is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy.

Determination of HER2 phosphorylation at tyrosine 1221/1222 improves prediction of poor survival for breast cancer patients with hormone receptor-positive tumors.

INTRODUCTION: High expression of total HER2 protein confers poor prognosis for breast cancer patients. HER2 is a member of the HER family consisting of four receptors, HER1 to HER4. HER receptor activity is regulated by a variety of mechanisms, and phosphorylation of the C-terminal part of the HER receptors is a marker for active signaling. The importance of phosphorylation and thereby activation of the HER1 to HER4 receptors, however, has not been investigated concomitantly in breast tumors. In the present study we examined the importance of active HER signaling in breast tumor biopsies and paired metastases, by evaluating the expression of phosphorylated HER1, HER2, HER3, Erk, Akt and the total level of HER4 and HER2. METHODS: Immunohistochemical analysis was performed on 268 primary breast tumors and 30 paired metastatic lesions from postmenopausal women with hormone receptor-positive breast tumors, who had received adjuvant tamoxifen therapy. The observed protein expression levels were analyzed for co-expression, for correlation to clinicopathological parameters and for prognostic value in relation to disease-free survival and overall survival. Lastly, the difference between protein levels in primary tumors versus metastasis was evaluated. RESULTS: In the primary tumors, 8%, 18%, 14% and 15% of cases were scored positive for total HER2, pHER1, pHER2 and pHER3 expression, respectively. HER4 was expressed with strong intensity in 68% and at moderate intensity in 29% of cases. The activated forms of Akt and Erk were quite uniformly expressed in the categories; negative, moderate or strong. In univariate analysis, expression of total HER2, pHER1, pHER2 and pHER3 was significantly associated with poor disease-free survival. Strong HER4 expression was associated with prolonged disease-free as well as with overall survival. Expression of pAkt and pErk was not correlated with survival. In multivariate analysis, pHER2 expression was clearly an independent marker for poor disease-free survival and overall survival when tested against tumor size, tumor grade, nodal status and HER2. Lastly, comparison of HER receptor expression in metastatic versus primary tumors showed a significant increase in expression of pHER1 and pHER3 in the metastases. CONCLUSIONS: In hormone receptor-positive breast cancer, determination of pHER2 yields additional prognostic information about poor prognosis compared with the current clinical standard for measuring HER2.

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes.

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10(-6) M) on DNA synthesis. When we added transforming growth factor-alpha at the time of plating, 10(-6) M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24-48 h after plating, 10(-6) M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10(-8) M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10(-6) M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.

Oligomers of ERBB3 have two distinct interfaces that differ in their sensitivity to disruption by heregulin.

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity.

Preclinical evaluation of targeted cytotoxic luteinizing hormone-releasing hormone analogue AN-152 in androgen-sensitive and insensitive prostate cancers.

PURPOSE AND EXPERIMENTAL DESIGN: To improve conventional chemotherapy, we developed cytotoxic analogues of luteinizing hormone-releasing hormone (LH-RH), which can be targeted to prostate cancers expressing LH-RH receptors. In view of pending clinical trials on cytotoxic LH-RH analogue AN-152, containing doxorubicin (DOX) linked to [D-Lys(6])-LH-RH, we investigated the effects of AN-152 on tumor growth of s.c. implanted androgen-sensitive LNCaP and MDA-PCa-2b prostate cancers, as well as androgen-independent C4-2 prostate cancers xenografted into the tibiae of nude mice. In the C4-2 study, serum prostate-specific antigen (PSA) levels were also measured. LH-RH receptors were analyzed by reverse transcription-PCR and ligand competition assay. We also evaluated whether AN-152 can affect mRNA expression of human epidermal growth factor receptor and HER-2 and -3 oncogenes RESULTS: After 32 days of treatment with AN-152, the growth of LNCaP cancers in castrated nude mice was strongly inhibited by 83% versus intact controls (P < 0.01) and 62% versus castrated controls (P < 0.05). In animals bearing MDA-PCa-2b prostate cancers, therapy with AN-152 for 25 days resulted in a 69% inhibition of tumor growth (P < 0.01 versus controls) and was more effective (P < 0.05) than equimolar doses of DOX or microcapsules of LH-RH agonist Decapeptyl. In nude mice bearing intraosseous C4-2 prostate cancers, treatment with AN-152 decreased serum PSA levels (P < 0.01) to 10.3 +/- 3.4 ng/ml from 24.8 +/- 4 ng/ml in controls, whereas DOX had no effect on PSA. The inhibitory effects of AN-152 on C4-2 tumors was accompanied by an increase in apoptosis and a decrease in tumor proliferation. Binding sites for LH-RH and the expression of mRNA for LH-RH receptors were found on s.c. C4-2 and MDA-PCa-2b tumors. The inhibition of MDA-PCa-2b tumors by AN-152 was associated with a significant decrease in mRNA expression for epidermal growth factor receptor, HER-2, and 3. CONCLUSIONS: Our findings suggest that cytotoxic analogue AN-152 could be considered for therapeutic trials in patients with advanced prostate carcinoma.

[Targeting epidermal growth factor receptor in cancer of the breast]. / Ciblage du récepteur du facteur de croissance épidermique dans les cancers du sein.

The erbB receptor family is part of the receptor tyrosine kinase superfamily and consists of four members erbB. The erbB receptor family has been shown to play an important role in both the development of the normal breast and the pathogenesis and progression of breast cancer. Receptor overexpression has also been shown to be a negative prognostic indicator and to correlate with both tumor invasiveness and a lack of responsiveness to standard treatment, both chemotherapy and hormonotherapy. The targeting of EGFR mainly resides in two approaches: tyrosine kinase inhibition and monoclonal antibodies blocking ligand fixation. Many experimental data support the potential role of targeting EGFR in breast cancer. Particularly tyrosine kinase inhibitors demonstrates activity as single agent or in association with hormonotherapy, chemotherapy and trastuzumab. The association of Iressa with hormonotherapy points out that theses agents may prevent or differ hormonoresistance. Moreover studies in situ carcinoma suggest that tyrosine kinase inhibitors may play a role in chemoprevention. So, targeting EGFR may be indicated in a large spectrum of breast tumors from early to advanced stages, hormone negative or positive breast tumors. However the complexity of erbB network requires the targeting of multiple molecular sites within the network and the characterization of tumor profiles in order to optimally select patients for these therapies.

Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95.

Neuregulin is a factor essential for synapse-specific transcription of acetylcholine receptor genes at the neuromuscular junction. Its receptors, ErbB receptor tyrosine kinases, are localized at the postjunctional membrane presumably to ensure localized signaling. However, the molecular mechanisms underlying synaptic localization of ErbBs are unknown. Our recent studies indicate that ErbB4 interacts with postsynaptic density (PSD)-95 (SAP90), a PDZ domain-containing protein that does not interact with ErbB2 or ErbB3. Using as bait the ErbB2 C terminus, we identified Erbin, another PDZ domain-containing protein that interacts specifically with ErbB2. Erbin is concentrated in postsynaptic membranes at the neuromuscular junction and in the central nervous system, where ErbB2 is concentrated. Expression of Erbin increases the amount of ErbB2 labeled by biotin in transfected cells, suggesting that Erbin is able to increase ErbB2 surface expression. Furthermore, we provide evidence that Erbin interacts with PSD-95 in both transfected cells and synaptosomes. Thus ErbB proteins can interact with a network of PDZ domain-containing proteins. This interaction may play an important role in regulation of neuregulin signaling and/or subcellular localization of ErbB proteins.