In vaccinated individuals serum bactericidal activity against B meningococci is abrogated by C5 inhibition but not by inhibition of the alternative complement pathway.

Introduction: Several diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci. Methods: Serum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodies against meningococcal outer membrane proteins. Results: While only a few pre-vaccination sera showed SBA against the tested B meningococcal isolates, 4CMenB vaccination induced potent complement-activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed factor H-binding protein (fHbp). SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, whereas alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on the SBA of post-4CMenB vaccination sera. Discussion: Compared to the SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele was remarkably resilient against the alternative pathway inhibition.

Inhibition of the different complement pathways has varying impacts on the serum bactericidal activity and opsonophagocytosis against Haemophilus influenzae type b.

Defense against Haemophilus influenzae type b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and complement inhibitors targeting the central component C3, the alternative pathway (AP; fB, fD), the lectin pathway (LP; MASP-2) and the terminal pathway (C5) on both effector functions. Findings may be relevant for the treatment of certain diseases caused by dysregulation of the complement system, where inhibitors of complement factors C3 or C5 are used. Inhibitors against other complement components are being evaluated as potential alternative treatment options that may carry a reduced risk of infection by encapsulated bacteria. Serum and reconstituted blood of healthy adults were tested for bactericidal activity before and after vaccination with the Hib capsule-conjugate vaccine ActHIB. Most sera had bactericidal activity prior to vaccination, but vaccination significantly enhanced SBA titers. Independently of the vaccination status, both C3 and C5 inhibition abrogated SBA, whereas inhibition of the LP had no effect. AP inhibition had a major inhibitory effect on SBA of pre- vaccination serum, but vaccination mitigated this inhibition for all disease isolates tested. Despite this, SBA-mediated killing of some Hib isolates remained retarded. Even for the most serum-resistant isolate, SBA was the dominating defense mechanism in reconstituted whole blood, as addition of blood cells to the serum did not enhance bacterial killing. Limited Fc receptor-mediated opsonophagocytosis was unmasked when bacterial killing by the membrane attack complex was blocked. In the presence of C3 or C5 inhibitors, addition of post-vaccination, but not of pre-vaccination serum to the blood cells triggered opsonophagocytosis, leading to suppression of bacterial multiplication. Taken together, our data indicate that for host defense against Hib, killing by SBA is more efficient than by blood cell opsonophagocytosis. However, additional defense mechanisms, such as bacterial clearance by spleen and liver, may play an important role in preventing Hib-mediated sepsis, in particular for Hib isolates with increased serum-resistance. Results indicate potentially improved safety profile of AP inhibitors over C3 and C5 inhibitors as alternative therapeutic agents in patients with increased susceptibility to Hib infection.

Blood Pressure Drops During Hospitalization for Acute Heart Failure Treated With Serelaxin: A Patient-Level Analysis of 4 Randomized Controlled Trials.

BACKGROUND: Hypotensive events and drops in systolic blood pressure (SBP-drop) are frequent in patients hospitalized with acute heart failure. We investigated whether SBP-drops are associated with outcomes in patients treated with serelaxin. METHODS: Patient-level retrospective analyses of 4 prospective trials investigating serelaxin in acute heart failure. Main inclusion criteria were SBP 125 to 180 mm Hg, pulmonary congestion, and elevated NT-proBNP (N-terminal pro-B-type natriuretic peptide). SBP-drops were prospectively defined as SBP<100 mm Hg, or, if SBP remained >100 mm Hg, a drop from baseline of 40 mm Hg from baseline. Outcomes were a short-term composite outcome (worsening heart failure, hospital readmission for heart failure or all-cause mortality through 14 days) and 180-day mortality. RESULTS: Overall, 2559/11 226 (23%) patients had an SBP-drop. SBP-drop, versus no SBP-drop, was associated with a worse outcome: cumulative incidence of 180-day mortality (11% versus 9%, hazard ratio [HR]. 1.21 [95% CI, 1.05-1.39]; P=0.009) and the short-term outcome (11% versus 9%, HR, 1.29 [95% CI, 1.13-1.49]; P<0.001). Of the 2 SBP-drop components, an SBP<100 mm Hg was associated with the worst outcome compared with a 40 mm Hg drop: short-term outcome (11% versus 10%) and HRs of 1.32 (95% CI, 1.13-1.55; P=0.0005) and 1.22 (95% CI, 0.97-1.56; P=0.09), for each component respectively, with a P value for interaction of 0.05. SBP-drops were associated with a worse short-term outcome in the placebo group (HR, 1.46 [95% CI, 1.19-1.79]; P=0.0003), but not in the serelaxin-group (HR, 1.18 [95% CI, 0.97-1.42]; P=0.10); P interaction=0.003. CONCLUSIONS: SBP-drops in patients with acute heart failure and normal to high SBP at admission is associated with worse short- and long-term outcomes especially for SBP <100 mm Hg. However, in patients treated with the intravenous vasodilator serelaxin, SBP-drops seemed less harmful. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifiers: NCT02064868, NCT02007720, NCT01870778, NCT00520806.

Alternative Complement Pathway Inhibition Does Not Abrogate Meningococcal Killing by Serum of Vaccinated Individuals.

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors.

Alternative Complement Pathway Inhibition Abrogates Pneumococcal Opsonophagocytosis in Vaccine-Naïve, but Not in Vaccinated Individuals.

To assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and PCV13-vaccinated subjects. With serotype 4 pneumococci as model, two different physiologic opsonophagocytosis assays based on either hirudin-anticoagulated whole blood or on washed cells from EDTA-anticoagulated blood reconstituted with active serum, were compared. Pneumococcal killing was measured in the presence of inhibitors targeting the complement components C3, C5, MASP-2, factor B or factor D. The two assay formats yielded highly consistent and comparable results. They highlighted the importance of alternative complement pathway activation for efficient opsonophagocytic killing in blood of vaccine-naïve subjects. In contrast, alternative complement pathway inhibition did not affect pneumococcal killing in PCV13-vaccinated individuals. Independent of amplification by the alternative pathway, even low capsule-specific antibody concentrations were sufficient to efficiently trigger classical pathway mediated opsonophagocytosis. In heat-inactivated or C3-inhibited serum, high concentrations of capsule-specific antibodies were required to trigger complement-independent opsonophagocytosis. Our findings suggest that treatment with alternative complement pathway inhibitors will increase susceptibility for invasive pneumococcal infection in non-immune subjects, but it will not impede pneumococcal clearance in vaccinated individuals.

Systolic Blood Pressure and Outcome in Patients Admitted With Acute Heart Failure: An Analysis of Individual Patient Data From 4 Randomized Clinical Trials.

BACKGROUND In acute heart failure (AHF), systolic blood pressure (SBP) is an important clinical variable. This study assessed the association between SBP and short-term and long-term outcomes in a large cohort of patients with AHF. METHODS AND RESULTS This is an analysis of 4 randomized controlled trials investigating serelaxin versus placebo in patients admitted with AHF and SBPs from 125 to 180 mm Hg. Outcomes were 180-day all-cause mortality and a composite end point of all-cause mortality, worsening heart failure, or hospital readmission for heart failure the first 14 days. Left ventricular ejection fraction (LVEF) was examined as LVEF<40% and LVEF≥40%. Multivariable Cox regression models were adjusted for known confounders of outcomes in AHF. A total of 10 533 patients with a mean age of 73 (±12) years and a mean SBP of 145 (±7) mm Hg were included. LVEF was assessed in 9863 patients (93%); 4737 patients (45%) had LVEF<40%. Increasing SBP was inversely associated with 180-day mortality (adjusted hazard ratio [HRadjusted], 0.93; 95% CI, 0.89-0.98; P=0.008 per 10 mm Hg increase) and with the composite end point (HRadjusted, 0.90; 95% CI, 0.85-0.94; P<0.001 per 10 mm Hg increase). A significant interaction with LVEF was observed, revealing that SBP was not associated with mortality in patients with LVEF≥40% (HRadjusted, 0.98; 95% CI, 0.91-1.04; per 10 mm Hg increase), but was strongly associated with increased mortality in LVEF<40% (HRadjusted, 0.84; 95% CI, 0.77-0.92; per 10 mm Hg increase). CONCLUSIONS Elevated SBP is associated with favorable short-term and long-term outcomes in patients with AHF. In our predefined subgroup analysis, we found that baseline SBP was not associated with mortality in LVEF≥40%, but was strongly associated with mortality in patients with LVEF<40%.

Cause of Death in Patients With Acute Heart Failure: Insights From RELAX-AHF-2.

OBJECTIVES: This study sought to better understand the discrepant results of 2 trials of serelaxin on acute heart failure (AHF) and short-term mortality after AHF by analyzing causes of death of patients in the RELAX-AHF-2 (Efficacy, Safety and Tolerability of Serelaxin When Added to Standard Therapy in AHF-2) trial. BACKGROUND: Patients with AHF continue to suffer significant short-term mortality, but limited systematic analyses of causes of death in this patient population are available. METHODS: Adjudicated cause of death of patients in RELAX-AHF-2, a randomized, double-blind, placebo-controlled trial of serelaxin in patients with AHF across the spectrum of ejection fraction (EF), was analyzed. RESULTS: By 180 days of follow-up, 11.5% of patients in RELAX-AHF-2 died, primarily due to heart failure (HF) (38% of all deaths). Unlike RELAX-AHF, there was no apparent effect of treatment with serelaxin on any category of cause of death. Older patients (≥75 years) had higher rates of mortality (14.2% vs. 8.8%) and noncardiovascular (CV) death (27% vs. 19%) compared to younger patients. Patients with preserved EF (≥50%) had lower rates of HF-related mortality (30% vs. 40%) but higher non-CV mortality (36% vs. 20%) compared to patients with reduced EF. CONCLUSIONS: Despite previous data suggesting benefit of serelaxin in AHF, treatment with serelaxin was not found to improve overall mortality or have an effect on any category of cause of death in RELAX-AHF-2. Careful adjudication of events in the serelaxin trials showed that older patients and those with preserved EF had fewer deaths from HF or sudden death and more deaths from other CV causes and from noncardiac causes. (Efficacy, Safety and Tolerability of Serelaxin When Added to Standard Therapy in AHF [RELAX-AHF-2]; NCT01870778).

Relationship between left ventricular ejection fraction and cardiovascular outcomes following hospitalization for heart failure: insights from the RELAX-AHF-2 trial.

AIMS: Although left ventricular ejection fraction (LVEF) is routinely used to categorize patients with heart failure (HF), whether it predicts outcomes after hospitalization for acute heart failure (AHF) is uncertain. Consequently, we assessed the relationship between LVEF and cardiovascular (CV) outcomes in a large, well characterized cohort of patients hospitalized for AHF. METHODS AND RESULTS: The 6128 patients from the RELAX-AHF-2 trial who had LVEF measured during AHF hospitalization were separated into LVEF quartiles and the relationship between LVEF and a composite of CV mortality and rehospitalization for HF or renal failure through 180 days was assessed. We found progressively lower risk for this composite outcome as LVEF increased (hazard ratio 0.95, 95% confidence interval 0.93-0.98 per 5% LVEF increase, P < 0.001) that was driven predominantly by decreased risk for rehospitalization. The smoothed spline curve depicting risk remained stable as LVEF decreased until reaching approximately 40%, at which point risk increased progressively with further reductions in LVEF. Significant differences between LVEF quartiles for post-discharge CV risk were seen in patients with an ischaemic aetiology or with a history of HF preceding index hospitalization, but were less robust in patients with non-ischaemic aetiology and absent in those with de novo HF. CONCLUSION: In patients hospitalized with AHF, CV events over 180 days were more frequent in patients with lower LVEF. This was due predominantly to a significant increase in risk for HF/renal failure rehospitalization but not in either CV or all-cause mortality. LVEF had greater prognostic value in patients with ischaemic aetiology or pre-existing HF.

Effects of Serelaxin in Patients with Acute Heart Failure.

BACKGROUND: Serelaxin is a recombinant form of human relaxin-2, a vasodilator hormone that contributes to cardiovascular and renal adaptations during pregnancy. Previous studies have suggested that treatment with serelaxin may result in relief of symptoms and in better outcomes in patients with acute heart failure. METHODS: In this multicenter, double-blind, placebo-controlled, event-driven trial, we enrolled patients who were hospitalized for acute heart failure and had dyspnea, vascular congestion on chest radiography, increased plasma concentrations of natriuretic peptides, mild-to-moderate renal insufficiency, and a systolic blood pressure of at least 125 mm Hg, and we randomly assigned them within 16 hours after presentation to receive either a 48-hour intravenous infusion of serelaxin (30 µg per kilogram of body weight per day) or placebo, in addition to standard care. The two primary end points were death from cardiovascular causes at 180 days and worsening heart failure at 5 days. RESULTS: A total of 6545 patients were included in the intention-to-treat analysis. At day 180, death from cardiovascular causes had occurred in 285 of the 3274 patients (8.7%) in the serelaxin group and in 290 of the 3271 patients (8.9%) in the placebo group (hazard ratio, 0.98; 95% confidence interval [CI], 0.83 to 1.15; P = 0.77). At day 5, worsening heart failure had occurred in 227 patients (6.9%) in the serelaxin group and in 252 (7.7%) in the placebo group (hazard ratio, 0.89; 95% CI, 0.75 to 1.07; P = 0.19). There were no significant differences between the groups in the incidence of death from any cause at 180 days, the incidence of death from cardiovascular causes or rehospitalization for heart failure or renal failure at 180 days, or the length of the index hospital stay. The incidence of adverse events was similar in the two groups. CONCLUSIONS: In this trial involving patients who were hospitalized for acute heart failure, an infusion of serelaxin did not result in a lower incidence of death from cardiovascular causes at 180 days or worsening heart failure at 5 days than placebo. (Funded by Novartis Pharma; RELAX-AHF-2 ClinicalTrials.gov number, NCT01870778.).

Evaluating the Efficacy, Safety, and Tolerability of Serelaxin When Added to Standard Therapy in Asian Patients With Acute Heart Failure: Design and Rationale of RELAX-AHF-ASIA Trial.

BACKGROUND: Acute heart failure (AHF), a common and growing health concern worldwide, is associated with high risk of post-discharge rehospitalization and mortality. Existing evidence indicates potential therapeutic benefits of serelaxin in Caucasian AHF patients, but corresponding data in Asians remain scarce. RELAX-AHF-ASIA, a multinational, randomized, double-blind, placebo-controlled, phase III trial, will evaluate the effects of serelaxin on symptom relief and clinical outcomes in Asian AHF patients, with the use of novel assessments. METHODS AND RESULTS: Patients with AHF, systolic blood pressure ≥125 mm Hg, and mild to moderate renal dysfunction will be randomized within 16 hours of presentation to receive 48-hour intravenous infusion of 30 µg ⋅ kg-1 ⋅ d-1 serelaxin or placebo in addition to standard therapy. The composite primary end point includes: (1) treatment success (moderate/marked improvement in patient-reported dyspnea and physician-assessed signs of congestion on day 2); (2) treatment failure (in-hospital worsening of signs and/or symptoms of heart failure [HF] requiring intensification of intravenous HF therapy or mechanical ventilation, renal/circulatory support, rehospitalization due to HF/renal-failure, or death through day 5); and (3) unchanged status. Secondary end points include time to in-hospital worsening HF through day 5 and all-cause and cardiovascular deaths through day 180. CONCLUSIONS: RELAX-AHF-ASIA, the largest randomized clinical trial in Asian AHF patients to date, has a novel composite primary end point and the potential to become a hallmark of AHF trials.

Blocking of FGFR signaling inhibits breast cancer cell proliferation through downregulation of D-type cyclins.

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation, we used a low molecular weight compound, PD173074, that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415, MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression, inhibition of cyclin D/cdk4 activity and, as a consequence, reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells, suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells, both MAPK and PI3K signaling pathways were suppressed. In conclusion, results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells.

ErbB receptors: directing key signaling networks throughout life.

The epidermal growth factor (EGF)-related peptides bind the ErbB receptors, inducing the formation of different homo- and heterodimers. Receptor dimerization promotes activation of the intrinsic kinase, leading to phosphorylation of specific tyrosines located in the ErbB's cytoplasmic region. These phosphorylated residues serve as docking sites for a variety of signaling molecules whose recruitment stimulates intracellular signaling cascades, which ultimately control diverse genetic programs. Particular ligand-receptor complexes have essential roles in embryonic development as well as in the adult. Finally, ErbB receptors are being pursued as therapeutic targets because aberrant ErbB activity has been observed in many human cancers. In this review, we discuss these data in more detail, illustrating the importance of tightly regulated ErbB signaling throughout life.

The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation.

ErbB2 is a receptor tyrosine kinase whose activity in normal cells depends on dimerization with another ligand-binding ErbB receptor. In contrast, amplification of c-erbB2 in tumors results in dramatic overexpression and constitutive activation of the receptor. Breast cancer cells overexpressing ErbB2 depend on its activity for proliferation, because treatment of these cells with ErbB2-specific antagonistic antibodies or kinase inhibitors blocks tumor cells in the G1 phase of the cell cycle. Intriguingly, loss of ErbB2 signaling is accompanied by a decrease in the phosphotyrosine content of ErbB3. On the basis of these results, it has been proposed that ErbB3 might be a partner for ErbB2 in promoting cellular transformation. To test this hypothesis and directly examine the role of the "kinase dead" ErbB3, we specifically ablated its expression with a designer transcription factor (E3). By infection of ErbB2-overexpressing breast cancer cells with a retrovirus expressing E3, we show that ErbB3 is an essential partner in the transformation process. Loss of functional ErbB2 or ErbB3 has similar effects on cell proliferation and cell cycle regulators. Furthermore, expression of constitutively active protein kinase B rescues the proliferative block induced as a consequence of loss of ErbB2 or ErbB3 signaling. These results demonstrate that ErbB2 overexpression and activity alone are insufficient to promote breast tumor cell division. Furthermore, we identify ErbB3's role, which is to couple active ErbB2 to the phosphatidylinositol 3-kinase/protein kinase B pathway. Thus, the ErbB2/ErbB3 dimer functions as an oncogenic unit to drive breast tumor cell proliferation.

The ErbB receptors and their role in cancer progression.

The involvement of the ErbB receptor tyrosine kinases in human cancer, as well as their essential role in a variety of physiological events during normal development, have motivated the interest in this receptor family. Approaches taken to block the activity of ErbB receptors in cancer cells have not only proven that they drive in vitro tumor cell proliferation, but have also become clinically relevant for targeting tumors with deregulated ErbB signaling. The mechanisms and downstream effectors through which the ErbB receptors influence processes linked to malignant development, including proliferation, cell survival, angiogenesis, migration, and invasion, are, however, only now becoming apparent. Our particular emphasis in this review will be on how ErbB receptors, in particular ErbB1 and ErbB2, contribute to processes linked to cancer progression. Importantly, in keeping with the emerging theme that ErbB receptors do not function in isolation, we will focus on receptor cooperativity, i.e., ErbB1 cooperates with other classes of receptors, and the ligand-less ErbB2 functions as a heterodimer with other ErbBs.

Wnt1 and Wnt5a induce cyclin D1 expression through ErbB1 transactivation in HC11 mammary epithelial cells.

Constitutive expression of Wnt1 and Wnt5a in HC11 mammary cells led to elevated TCF transcriptional activity. Intriguingly, Wnt-expressing cells also displayed activation of ErbB1 and mitogen-activated protein kinase (MAPK), in contrast to control HC11 cells, which did not. Furthermore, conditioned media harvested from Wnt-expressing cells stimulated ErbB1 and the MAPK cascade when added to control cells. This process was rapid and could be blocked by an ErbB1 antibody that interferes with ligand binding and by matrix metalloproteinase (MMP) inhibitors. These results suggest that in mammary cells Wnt binding to its receptor, Frizzled (Fz), transactivates ErbB1, probably by MMP-mediated release of soluble ErbB1 ligands. Importantly, Wnt-transactivated ErbB1 was responsible for MAPK activation and the increased levels of cyclin D1 present in the Wnt-expressing HC11 cells. Our finding that Wnts transactivate ErbB1 in addition to stimulating the prototypic beta-catenin/TCF pathway may help to explain why wnt1 is a potent oncogene in the mammary gland.

Distinct roles for phosphoinositide 3-kinase, mitogen-activated protein kinase and p38 MAPK in mediating cell cycle progression of breast cancer cells.

Addition of the ErbB-ligand, Heregulinbeta1 (HRG), to breast tumour-derived T47D cells promotes D-cyclin expression, p21(cip1) synthesis, cyclin-dependent kinase (CDK) activation through re-distribution of p27(kip1) and DNA synthesis. In contrast EGF has no effect on T47D cell cycle progression. By comparing these two ligands and the use of specific inhibitors for phosphatidylinositol-3 kinase (PI3K), mitogen-activated protein kinase (MAPK) and p38MAPK, we have identified several molecular mechanisms required for ErbB receptor-mediated proliferation. The PI3K, MAPK and p38MAPK pathways each displayed distinct activation profiles in response to either HRG or EGF, with obvious differences in both the intensity and duration of signal output. Through inhibition of each of these pathways it is apparent that each pathway is necessary, yet insufficient alone, to stimulate proliferation. Each pathway regulates distinct subsets of essential cell cycle regulators and integration of these signal networks is required for the timely expression of these components, which culminates in cell cycle progression. Significantly, the mechanisms controlling ligand-stimulated proliferation through ErbB2 are strikingly similar to the mechanisms through which overexpressed, constitutively activated, ErbB2 orchestrates uncontrolled proliferation in cancer cells. This suggests that downstream effectors of ErbB receptors represent good therapeutic targets for breast cancer.