Transactivation Of ErbB2 Research Articles

Abstract A119: Antibody blockade of Semaphorin 4D neutralizes barrier to immune infiltration and facilitates immune-mediated tumor rejection

Abstract Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, we describe a novel immunomodulatory function of SEMA4D in regulation of immune cell infiltration and activity in the tumor microenvironment (TME). Purpose: Characterize immune-related and anti-tumor activity mediated by antibody neutralization of SEMA4D, as a single agent and in combination with other immunomodulatory therapies. Methods: Blockade of SEMA4D with monoclonal murine antibody was evaluated in subcutaneous models, as well as an orthotopic ERBB2+ breast carcinoma syngeneic model. Anti-tumor immune response in pre-clinical models was characterized by selective in vivo immune cell depletions, as well as immunohistochemistry, flow cytometry, and functional assays. The safety and tolerability of a humanized anti-SEMA4D antibody VX15/2503 was assessed in Phase I clinical trials in oncology. Results: SEMA4D restricts migration of macrophage cell lines in vitro. Strong expression of SEMA4D at the invasive margins of actively growing in vivo tumors modulates the infiltration and spatial distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient and facilitates recruitment of potent antigen presenting cells and T lymphocytes into the TME, shifting the balance of cytokines toward increased Th1 and reduced immunosuppressive cytokines. This orchestrated change in the tumor architecture was associated with durable tumor rejection and immunologic memory in preclinical models. Immune-mediated tumor rejection may enhance the disruption of ERBB2 transactivation with SEMA4D receptors, which has been reported for ERBB2 and Met oncogenes. Importantly, the immunomodulatory activity of anti-SEMA4D antibody can also be further enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival, with significant 58% increase in tumor regression and maximal increase in survival, as compared to monotherapy. Treatment with anti-SEMA4D antibodies was well tolerated in nonclinical and clinical studies, including completion of a Phase I prospective multiple ascending dose trial in patients with advanced refractory solid tumors. Weekly doses of between 0.3 and 20 mg/kg were administered; no MTD was determined. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. Progression free survival correlated with elevated baseline lymphocyte counts, supporting an immune mediated mechanism of action for VX15/2503. Conclusion: Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. A phase 1b/2a trial of combination therapy with immune checkpoint inhibition is planned. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Crystal Mallow, Laurie A. Winters, Christine Reilly, Ekaterina Klimatcheva, Janaki Veeraraghavan, Alan S. Jonason, Maria Scrivens, Renee Kirk, Alan Howell, Leslie Balch, John E. Leonard, Mark Paris, Terrence L. Fisher, Ernest S. Smith, Maurice Zauderer. Antibody blockade of Semaphorin 4D neutralizes barrier to immune infiltration and facilitates immune-mediated tumor rejection. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A119.

Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor.

Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1–7) [Ang-(1–7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1–7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1–7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1–7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1–7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1–7). Ang-(1–7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1–7) that could be prevented by D-Pro7-Ang-(1–7) in VSMC. These data suggest that Ang-(1–7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1–7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications.

Abstract 278: Antibody blockade of semaphorin 4D promotes infiltration of activated tumor infiltrating leukocytes and reverses tumor growth

Abstract Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, effects on cellular movement mediated by SEMA4D/PLXNB1 interactions have been described in relation to vascular stabilization and transactivation of ERBB2. Herein, we describe a novel function of SEMA4D in regulation of immune cell infiltration and activity in the tumor microenvironment (TME). PURPOSE: Characterize immune-related and anti-tumor activity mediated by antibody neutralization of SEMA4D, as a single agent or in combination with other immunomodulatory therapies. METHODS: Blockade of SEMA4D with monoclonal murine antibody was evaluated in subcutaneous Colon26 and orthotopic ERBB2+ breast carcinoma syngeneic models. Growth kinetics were assessed following selective in vivo immune cell depletions. Anti-tumor immune response was characterized by immunohistochemistry, FACS, and functional assays. RESULTS: Strong expression of SEMA4D at the invasive margins of actively growing tumors modulates the infiltration and spatial distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient and enhances recruitment of activated antigen presenting cells and lymphocytes into the TME, shifting the balance of cytokines toward increased Th1 and reduced immunosuppressive cytokines. This orchestrated change in the tumor architecture was associated with durable tumor rejection and immunologic memory in Colon26 and ERBB2+ mammary carcinoma models. Efficacy was dependent on both active adaptive and innate immune cell responses. The immunomodulatory activity of anti-SEMA4D antibody can be enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival, with significant 58% increase in tumor regression and >295% increase in survival, as compared to monotherapy. CONCLUSION: Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. Humanized anti-SEMA4D therapeutic antibody, VX15/2503, has completed a Phase I prospective multiple ascending dose trial in 42 adult patients with advanced refractory solid tumors, in which the highest doses were well tolerated. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. A phase 1b/2a trial of combination therapy is planned. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Crystal Mallow, Laurie A, Winters, Christine Reilly, Katya Klimatcheva, Janaki Veeraraghavan, Alan S. Jonason, Maria Scrivens, Renee Kirk, Sue Giralico, Alan Howell, John E. Leonard, Mark Paris, Terrence L. Fisher, Ernest S. Smith, Maurice Zauderer. Antibody blockade of semaphorin 4D promotes infiltration of activated tumor infiltrating leukocytes and reverses tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 278. doi:10.1158/1538-7445.AM2015-278

Antibody Blockade of Semaphorin 4D Promotes Immune Infiltration into Tumor and Enhances Response to Other Immunomodulatory Therapies

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 (PLXNB1) are broadly expressed in murine and human tumors, and their expression has been shown to correlate with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, SEMA4D-PLXNB1 interactions have been reported to affect vascular stabilization and transactivation of ERBB2, but effects on immune-cell trafficking in the tumor microenvironment (TME) have not been investigated. We describe a novel immunomodulatory function of SEMA4D, whereby strong expression of SEMA4D at the invasive margins of actively growing tumors influences the infiltration and distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient of expression, enhances recruitment of activated monocytes and lymphocytes into the tumor, and shifts the balance of cells and cytokines toward a proinflammatory and antitumor milieu within the TME. This orchestrated change in the tumor architecture was associated with durable tumor rejection in murine Colon26 and ERBB2(+) mammary carcinoma models. The immunomodulatory activity of anti-SEMA4D antibody can be enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival. Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the TME and inhibit tumor progression.

β4 Integrin signaling induces expansion of prostate tumor progenitors.

The contextual signals that regulate the expansion of prostate tumor progenitor cells are poorly defined. We found that a significant fraction of advanced human prostate cancers and castration-resistant metastases express high levels of the β4 integrin, which binds to laminin-5. Targeted deletion of the signaling domain of β4 inhibited prostate tumor growth and progression in response to loss of p53 and Rb function in a mouse model of prostate cancer (PB-TAg mice). Additionally, it suppressed Pten loss-driven prostate tumorigenesis in tissue recombination experiments. We traced this defect back to an inability of signaling-defective β4 to sustain self-renewal of putative cancer stem cells in vitro and proliferation of transit-amplifying cells in vivo. Mechanistic studies indicated that mutant β4 fails to promote transactivation of ErbB2 and c-Met in prostate tumor progenitor cells and human cancer cell lines. Pharmacological inhibition of ErbB2 and c-Met reduced the ability of prostate tumor progenitor cells to undergo self-renewal in vitro. Finally, we found that β4 is often coexpressed with c-Met and ErbB2 in human prostate cancers and that combined pharmacological inhibition of these receptor tyrosine kinases exerts antitumor activity in a mouse xenograft model. These findings indicate that the β4 integrin promotes prostate tumorigenesis by amplifying ErbB2 and c-Met signaling in tumor progenitor cells.

Abstract 611: Etanercept as a new tool for treatment of Herceptin-resistant breast cancer induced by tumor necrosis factor alpha

Abstract Breast cancer overexpressing ErbB2 is associated with high aggressiveness and elevated metastatic potential. Several therapies have been designed targeting receptor tyrosine kinase ErbB2, such as the low molecular weight inhibitor Lapatinib and the monoclonal antibody Herceptin. We have already demonstrated that tumor necrosis factor alpha (TNF) induces proliferation of BT-474 and SKBR-3 human and C4HD murine breast cancer cells which overexpress ErbB2, through the transactivation of ErbB2 and subsequent activation of transcription factor NF-κB, and through increase of Cyclin D1 protein expression. In the present study we evaluated the effectiveness of clinical drugs which block ErbB2, in order to inhibit NF-κB transcriptional activity and TNF-induced proliferation. We transfected BT-474 and SKBR-3 breast cancer cells with a luciferase reporter gene under control of a NF-κB response element and observed that the pharmacological ErbB2 inhibitor AG825 (100 μM)and the Lapatinib analog GW2974 (1 μM) blocked NF-κB activity induced by TNF. However, when we used 10 μg/ml Herceptin, TNF still activated NF-κB. We also monitored NF-κB activation through Western blot of phosphorylated IκBα, the protein which inhibits NF-κB traslocation to the nucleus, with same results. By reporter gene assays, we then explored activation of Cyclin D1 promoter, a target gene of NF-κB. While both AG825 and GW2974 blocked TNF-induced Cyclin D1 promoter activation, Herceptin did not. The endogenous protein Cyclin D1 mirrored the profile obtained with reporter gene assays. BT-474 proliferation increased in the presence of TNF (75 ± 12% vs control cells) measured by 3H-thimidine incorporation, cell count and flow cytometry of cells stained with propidium iodide. AG825 and GW2974 completely blocked TNF-induced proliferation, but Herceptin failed to do so. Bearing in mind that TNF is frequently expressed in invasive breast tumors, our results suggest that TNF may be one of the factors which confer Herceptin resistance. We therefore hypothesized that blocking TNF through the TNFR2-FcIgG fusion protein Etanercept may overcome Herceptin resistance. We observed that Etanercept (5 mg/kg, i.p. twice wk) inhibited TNF-induced in vitro proliferation of C4HD cells, a murine breast adenocarcinoma that produces TNF. When this tumor was growth in nude mice and treated with Etanercept the tumor size was reduced by 37.6 ± 1.1 % (P<0.05). Interestingly, histological examination of said tumors revealed that there were fewer mitotic figures and a lower percentage of necrotic and fibrotic areas in the tumors from mice treated with Etanercept, as compared to the group treated with an irrelevant IgG. Our results propose Etanercept as a new agent to overcome clinically observed Herceptin resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 611.

Transactivation of ErbB-2 induced by tumor necrosis factor α promotes NF-κB activation and breast cancer cell proliferation

Tumor necrosis factor alpha (TNFalpha) is a pleiotropic cytokine which, acting locally, induces tumor growth. Accumulating evidence, including our findings, showed that TNFalpha is mitogenic in breast cancer cells in vitro and in vivo. In the present study, we explored TNFalpha involvement on highly aggressive ErbB-2-overexpressing breast cancer cells. We found that TNFalpha induces ErbB-2 phosphorylation in mouse breast cancer C4HD cells and in the human breast cancer cell lines SK-BR-3 and BT-474. ErbB-2 phosphorylation at Tyr877 residue was mediated by TNFalpha-induced c-Src activation. Moreover, TNFalpha promoted ErbB-2/ErbB-3 heterocomplex formation, Akt activation and NF-kappaB transcriptional activation. Inhibition of ErbB-2 by addition of AG825, an epidermal growth factor receptor/ErbB-2-tyrosine kinase inhibitor, or knockdown of ErbB-2 by RNA interference strategy, blocked TNFalpha-induced NF-kappaB activation and proliferation. However, the humanized monoclonal antibody anti-ErbB-2 Herceptin could not inhibit TNFalpha ability to promote breast cancer growth. Interestingly, our work disclosed that TNFalpha is able to transactivate ErbB-2 and use it as an obligatory downstream signaling molecule in the generation of mitogenic signals. As TNFalpha has been shown to be present in the tumor microenvironment of a significant proportion of human infiltrating breast cancers, our findings would have clinical implication in ErbB-2-positive breast cancer treatment.

Aberrant expression of proteinase‐activated receptor 4 promotes colon cancer cell proliferation through a persistent signaling that involves Src and ErbB‐2 kinase

Thrombin is now recognized as an important factor in many cancers. Here, we examined the expression and role of the recently discovered thrombin receptor PAR4, in human colon cancer cells. PAR4 mRNA was found in 10 out of 14 (71%) human colon cancer cell lines tested but not in epithelial cells isolated from normal human colon. This finding is in line with immunostaining results of PAR4 in human colon tumors and its absence in normal human colonic mucosa. Investigation of the functional significance of the aberrant expression of PAR4 in colon cancer cells revealed (i) a prompt increase in intracellular calcium concentration on challenge with PAR4-specific agonist AP4 (100 microM) and (ii) marked mitogenic response (2.5-fold increase in cell number) in a dose-dependent manner on treatment with AP4 (0.1-300 microM). Analysis of the signaling pathways downstream of PAR4 activation in HT29 cells showed (i) a sustained phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) and (ii) the involvement of epidermal growth factor receptor B-2 (ErbB-2) but not of epidermal growth factor receptor in PAR4-induced mitogen-activated protein kinase activation. Tyrphostin AG1478, the ErbB inhibitor, reversed the action of AP4 on ERK1/2 and ErbB-2 phosphorylation and HT29 cell growth. Finally, the Src inhibitor PP2 abrogated ErbB-2 and ERK phosphorylation and HT29 cell proliferation, suggesting the essential role of Src activity in PAR4-induced phosphorylation of ErbB-2. These data highlight the role of PAR4 as a new important player in the control of colon tumors and underline the critical role of ErbB-2 transactivation.

Leptin signaling in breast cancer: An overview

The adipocyte-derived peptide leptin acts through binding to specific membrane receptors, of which six isoforms (obRa-f) have been identified up to now. Binding of leptin to its receptor induces activation of different signaling pathways, including the JAK/STAT, MAPK, IRS1, and SOCS3 signaling pathways. Since the circulating levels of leptin are elevated in obese individuals, and excess body weight has been shown to increase breast cancer risk in postmenopausal women, several studies addressed the role of leptin in breast cancer. Expression of leptin and its receptors has been demonstrated to occur in breast cancer cell lines and in human primary breast carcinoma. Leptin is able to induce the growth of breast cancer cells through activation of the Jak/STAT3, ERK1/2, and/or PI3K pathways, and can mediate angiogenesis by inducing the expression of vascular endothelial growth factor (VEGF). In addition, leptin induces transactivation of ErbB-2, and interacts in triple negative breast cancer cells with insulin like growth factor-1 (IGF-1) to transactivate the epidermal growth factor receptor (EGFR), thus promoting invasion and migration. Leptin can also affect the growth of estrogen receptor (ER)-positive breast cancer cells, by stimulating aromatase expression and thereby increasing estrogen levels through the aromatization of androgens, and by inducing MAPK-dependent activation of ER. Taken together, these findings suggest that the leptin system might play an important role in breast cancer pathogenesis and progression, and that it might represent a novel target for therapeutic intervention in breast cancer.

Persistent transactivation of EGFR and ErbB2/HER2 by protease-activated receptor-1 promotes breast carcinoma cell invasion

Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signal-regulated kinase-1 and -2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Galpha(i/o) and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.

Macrophage inhibitory cytokine-1 activates AKT and ERK-1/2 via the transactivation of ErbB2 in human breast and gastric cancer cells

Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor-beta superfamily, which is overexpressed in a variety of human cancers, including breast and gastric cancer. The function of MIC-1 in cancer remains controversial and its signaling pathways remain poorly understood. In this study, we demonstrate that MIC-1 induces the transactivation of ErbB2 in SK-BR-3 breast and SNU-216 gastric cancer cells. MIC-1 induced a significant phosphorylation of Akt and ERK-1/2, and also effected an increase in the levels of tyrosine phosphorylation of ErbB1, ErbB2 and ErbB3 in SK-BR-3 and SNU-216 cells. The treatment of these cells with AG825 and AG1478, inhibitors specific for ErbB2 tyrosine kinase, resulted in the complete abolition of MIC-1-induced Akt and ERK-1/2 phosphorylation. Furthermore, the small-interfering RNA-mediated downregulation of ErbB2 significantly reduced not only the phosphorylation of Akt and ERK-1/2 but also the invasiveness of the cells induced by MIC-1. Our results show that ErbB2 activation performs a crucial function in MIC-1-induced signaling pathways. Further investigations revealed that MIC-1 induced the expression of the hypoxia inducible factor-1alpha protein and the expression of its target genes, including vascular endothelial growth factor, via the activation of the mammalian target of rapamycin (mTOR) signaling pathway. Stimulation of SK-BR-3 with MIC-1 profoundly induces the phosphorylation of mTOR and its downstream substrates, including p70S6K and 4E-BP1. Collectively, these results show that MIC-1 may participate in the malignant progression of certain human cancer cells that overexpress ErbB2 through the transactivation of ErbB2 tyrosine kinase.

Tyrphostin ErbB2 Inhibitors AG825 and AG879 Have Non-specific Suppressive Effects on gp130/ STAT3 Signaling

Although the interaction between gp130 and the ErbB family has frequently been shown in cancer cells, the mechanism of this interaction remains unclear and controversial. In the present study, we found that specific tyrphostin inhibitors of ErbB2 (AG825 and AG879), but not ErbB1 inhibitor (AG1478), suppressed IL-6-induced tyrosine phosphorylation of STAT3 in schwannoma cells. However, biochemical evidence for transactivation of ErbB2 by IL-6 was not observed. Additionally, the inhibition of ErbB2 expression, with either a specific RNAi or transfection of an ErbB2 mutant lacking the intracellular domain did not inhibit the IL-6-induced tyrosine phosphorylation of STAT3. Thus, it seems that tyrphostins, which are known as specific inhibitors of the ErbB2 kinase, may have non-specific suppressive effects on the IL-6/STAT3 pathway.

Transactivation of erbB2 by short and long isoforms of leptin receptors

Transactivation of erbB2 by short and long isoforms of leptin receptors

Transactivation of erbB2 by short and long isoforms of leptin receptors

We generated kinase-positive and kinase-negative erbB2 tagged with YFP and the long form of leptin receptor (LEPRb) tagged with CFP. Both were as active as their untagged analogs. Both short and long isoforms of leptin receptor phosphorylated and thereby activated erbB2 upon leptin binding and enhanced MAPK activity. Our results unveil a novel route by which leptin may provoke erbB2's phosphorylation and thus enhance its oncogenic potential independently of HER family ligands or its overexpression. Using FRET technology in living cells, we found no evidence of complex formation between erbB2 and prolactin or leptin receptors, indicating that the transactivation occurs through an indirect interaction.

Identification of a Region within the ErbB2/HER2 Intracellular Domain That Is Necessary for Ligand-independent Association

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads to erbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966-968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association.

Transactivation of ErbB2 and ErbB3 by Tumor Necrosis Factor-α and Anisomycin Leads to Impaired Insulin Signaling through Serine/Threonine Phosphorylation of IRS Proteins

The cellular pathways involved in the impairment of insulin signaling by cellular stress, triggered by the inflammatory cytokine tumor necrosis factor-alpha (TNF) or by translational inhibitors like cycloheximide and anisomycin were studied. Similar to TNF, cycloheximide and anisomycin stimulated serine phosphorylation of IRS-1 and IRS-2, reduced their ability to interact with the insulin receptor, inhibited the insulin-induced tyrosine phosphorylation of IRS proteins, and diminished their association with phosphatidylinositol 3-kinase (PI3K). These defects were partially reversed by wortmannin and LY294002, indicating that a PI3K-regulated step is critical for the impairment of insulin signaling by cellular stress. Induction of cellular stress resulted in complex formation between PI3K and ErbB2/ErbB3 and enhanced PI3K activity, implicating ErbB proteins as downstream effectors of stress-induced insulin resistance. Indeed, stimulation of ErbB2/ErbB3 by NDFbeta1, the ErbB3 ligand, inhibited IRS protein tyrosine phosphorylation and recruitment of downstream effectors. Specific inhibitors of the ErbB2 tyrosine kinase abrogated the activation of ErbB2/ErbB3 and in parallel prevented the reduction in IRS protein functions. Taken together, our results suggest a novel mechanism by which cellular stress induces cross-talk between two different signaling pathways. Stress-dependent transactivation of ErbB2/ErbB3 receptors triggers a PI3K cascade that induces serine phosphorylation of IRS proteins culminating in insulin resistance.

Structural requirements for ErbB2 transactivation

ErbB2 is a unique member of the ErbB family of receptor tyrosine kinases that is distinguished by the fact that no ligand has yet been identified. Due to the absence of an ErbB2 ligand, alternative mechanisms are used for ErbB2 activation. As such, when ErbB2 is over-expressed, kinase activation occurs in the absence of ligand because of constitutive homodimerization. However, at normal expression levels ErbB2 acts as the shared coreceptor for the ErbB family, and these heterodimeric complexes are activated in response to the partner ligand. While the extracellular domain and transmembrane domains are necessary for ErbB2 transactivation, the carboxy terminus is also required. Specifically, ligand-dependent ErbB2 transactivation requires a discrete three-amino-acid segment, located at the C-terminus of ErbB family members ErbB3, ErbB4, and the epidermal growth factor receptor. Transactivation of ErbB2 via the three-amino-acid segment likely represents a conserved mechanism for regulated signaling by the ErbB family of receptors.

Stimulation of Mitogenic Pathways through Kinase-Impaired Mutants of the Epidermal Growth Factor Receptor

Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the γ-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [3H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [3H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [3H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.

Molecular requirements for the effect of neuregulin on cell spreading, motility and colony organization.

Neuregulin can trigger morphogenetic signals in cells both in vivo and in culture through the activation of receptors from the ErbB family. We have ectopically expressed various ErbB-receptors in 32D myeloid cells lacking endogenous ErbB-proteins, and in CHO cells, which express only ErbB-2. We show here that activation of ErbB-3/ErbB-2 heterodimeric receptors triggers PI3-kinase-dependent lamellipodia formation and spreading, while individual ErbB-receptor homodimers as well as ErbB-3/ErbB-1 heterodimers are much less effective. CHO cells expressing ErB-3/ErbB-2 together with N-cadherin, an adhesion receptor, form epithelioid colonies. Neuregulin activates cell motility leading to transition of these colonies into ring-shaped multicellular arrays, similar to those induced by neuregulin in epithelial cells of different types (Chausovsky et al., 1998). This process requires both PI3-kinase and MAP kinase kinase activity and depends on coordinated changes in the actin- and microtubule-based cytoskeleton. Transactivation of ErbB-2 is not sufficient for the activation of cell motility and ring formation, and the C-terminal domain of ErbB-3 bearing the docking sites for the p85 subunit of PI3-kinase is essential for these morphogenetic effects. Thus, ErbB-3 in conjunction with ErbB-2 mediates, via its C-terminal domain, cytoskeletal and adhesion alterations which activate cell spreading and motility, leading to the formation of complex structures such as multicellular rings. Oncogene (2000) 19, 878 - 888.

NDF/heregulin-induced cell cycle changes and apoptosis in breast tumour cells: role of PI3 kinase and p38 MAP kinase pathways.

Neu differentiation factor (NDF)/heregulin activates ErbB2 via heterodimerization with the NDF receptors ErbB3 and ErbB4. Cells which express normal levels of these receptors are often growth stimulated by NDF, whereas SKBR3, and other ErbB2-overexpressing breast tumour cells are growth inhibited. We demonstrate here that in SKBR3 cells, NDF induces G1 progression but also causes a G2 delay from day 1 and apoptosis from days 2-3. G1 progression was associated with ErbB2 transactivation of ErbB3 and subsequent stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway whereas apoptosis was dependent on p38 MAPK. Inhibition of ERK1/ERK2 had no effect on cell cycle progression or apoptosis. Activation of ErbB3 and PI3K was also seen with betacellulin (BTC) but not epidermal growth factor (EGF) and correlated with the growth effects of these ligands. All three ligands induced short-term activation of p38 MAPK in a c-Src-dependent manner. However, only NDF caused a second, c-Src-independent increase in p38 MAPK activity which was required for apoptosis.