Autocrine Growth Loop of The Epidermal Growth Factor Receptor in Normal and Immortalized Human Bronchial Epithelial Cells

Epidermal growth factor (EGF) is a potent growth factor for human normal bronchial epithelial (HBE) cells and lung cancer cells, which often demonstrate an EGF receptor (EGFR) autocrine loop. We have found that HBE cells are capable of proliferating in basal medium without EGF supplementation, and this suggests the probable presence of an active EGFR autocrine loop in non-neoplastic HBE cells. Northern blot hybridization shows that the parental and immortalized HBE cells express comparable and high levels of mRNA for EGFR, transforming growth factor-alpha (TGF-α), and amphiregulin (AR), but not EGF. Incubation with neutralizing monoclonal antibodies (mAb) against EGFR partially inhibits the growth of these cells. Immunohistochemistry shows that HBE cells express the TGF-α peptidein vitroandin vivo,however, neutralizing mAbs against TGF-α fail to inhibit their proliferation. In contrast, AR stimulates the growth of HBE cells. Thus, several EGF-family ligands appear to be involved functionally in the EGFR autocrine growth loop in HBE cells.     

Expression and function of amphiregulin during murine preimplantation development

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-α significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-α, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-α in these embryos. Mol. Reprod. Dev. 47:271–283, 1997. © 1997 Wiley-Liss, Inc.     

PKC�� take part in CCR7/NF-��B autocrine signaling loop in CCR7-positive squamous cell carcinoma of head and neck

Metastatic squamous cell carcinoma of head and neck (SCCHN) has been shown to express chemokine receptor 7 (CCR7). The role of nuclear factor-κB (NF-κB) in propagating an autocrine signaling loop in CCR7-positive SCCHN cells may provide a clinically useful biomarker of disease status and response to therapy. In this article, we hypothesized that PKCα might be involved in the CCR7/NF-κB autocrine signaling loop. Results showed that CCL19 induced the activation of PKCα, and the increased activity of PKCα was abolished by CCR7 mAb. PKCα inhibition with G?6976 led to significant reduction in the activation and nuclear translocation of NF-κB induced by CCL19. Immunohistochemical assay also showed that CCR7 and PKCα were highly expressed in SCCHN and correlated with each other, which was significantly related to lymph node metastasis and clinical stage. Taken together, PKCα is involved in the CCR7/NF-κB autocrine signaling loop.     

EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells

Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met^flx/flx and Met^−/− oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-β)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met^flx/flx cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met^flx/flx and Met^−/− oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in normal, untransformed oval cells, c-Met and EGFR represent critical molecular players to control proliferation and survival that function independent of one another.     

Cyclic phosphatidic acid influences the expression and regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 cells

The abundance of transforming growth factor-beta (TGF-β) in normal airway epithelium suggests its participation in physiological processes to maintain airway homeostasis. The current study was designed to address the hypothesis that TGF-β1 and TGF-β2 might contribute to normal reparative response of airway epithelial cells (AECs). Treatments with exogenous TGF-β1 or TGF-β2 significantly enhanced wound repair of confluent AEC monolayers. Mechanical injury of AEC monolayers induced production of both TGF-β1 and TGF-β2. Wound repair of AECs was significantly reduced by a specific inhibitor of TGF-β type I receptor kinase activity. We investigated whether the TGF-β-enhanced repair required epidermal growth factor receptor (EGFR) transactivation and secretion of EGFR ligands. Both TGF-β1 and TGF-β2 enhanced EGFR phosphorylation and induced production of heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-α) in AECs. Moreover, treatment with a broad-spectrum metalloproteinase inhibitor or anti-HB-EGF and anti-TGF-α antibodies inhibited the wound repair and the EGFR phosphorylation by TGF-β1 and TGF-β2, indicating that the TGF-β1 and TGF-β2 effects on wound repair required the release of HB-EGF and TGF-α. Our data, for the first time, have shown that both TGF-β1 and TGF-β2 play a stimulatory role in airway epithelial repair through EGFR phosphorylation following autocrine production of HB-EGF and TGF-α. These findings highlight an important collaborative mechanism between TGF-β and EGFR in maintaining airway epithelial homeostasis.     

Rap1 mediates galanin receptor 2-induced proliferation and survival in squamous cell carcinoma

Previously we showed that galanin, a neuropeptide, is secreted by human squamous cell carcinoma of the head and neck (SCCHN) in which it exhibits an autocrine mitogenic effect. We also showed that rap1, a ras-like signaling protein, is a critical mediator of SCCHN progression. Given the emerging importance of the galanin cascade in regulating proliferation and survival, we investigated the effect of GAL on SCCHN progression via induction of galanin receptor 2 (GALR2)-mediated rap1 activation. Studies were performed in multiple SCCHN cell lines by inducing endogenous GALR2, by stably overexpressing GALR2 and by downregulating endogenous GALR2 with siGALR2. Cell proliferation and survival, mediated by the ERK and AKT signaling cascades, respectively, were evaluated by functional and immunoblot analysis. The role of rap1 in GALR2-mediated proliferation and survival was evaluated by modulating expression. Finally, the effect of GALR2 on tumor growth was determined. GALR2 stimulated proliferation and survival via ERK and AKT activation, respectively. Knockdown or inactivation of rap1 inhibited GALR2-induced, AKT and ERK-mediated survival and proliferation. Overexpression of GALR2 promoted tumor growth in vivo. GALR2 promotes proliferation and survival in vitro, and promotes tumor growth in vivo, consistent with an oncogenic role for GALR2 in SCCHN.     

Nimotuzumab,a promising therapeutic monoclonal for treatment of tumors of epithelial origin

Nimotuzumab is a humanized therapeutic monoclonal antibody against epidermal growth factor receptor (EGFR). Clinical trials are ongoing globally to evaluate nimotuzumab in different indications. Nimotuzumab has been granted approval for use in squamous cell carcinoma of head and neck (SCCHN), glioma and nasopharyngeal cancer in different countries. This review focuses on the unique functional characteristics of nimotuzumab. Also, it discusses the safety and efficacy data obtained from the Phase IIb clinical trial conducted in India in SCCHN. Post marketing surveillance data from Cuba for the use of nimotuzumab in pediatric and adult glioma is also discussed. Overall, nimotuzumab has immense therapeutic potential in cancers of epithelial origin.Key words: nimotuzumab, EGFR, humanized, monoclonal antibody, SCCHN, glioma, overall survival     

Involvement of cyclooxygenase-2 in proliferation and morphogenesis induced by transforming growth factor alpha in gastric epithelial cells

Transforming growth factor alpha is one of the most potent growth factors for gastrointestinal epithelium. In this study, we examined the roles of cyclooxygenase-2 on proliferation and morphogenesis of RGM1 rat gastric epithelial cells after stimulation with transforming growth factor alpha in vitro, RGM1 cells increased expression of cyclooxygenase-2 messenger RNA 20-60 min after stimulation with transforming growth factor alpha. Transforming growth factor alpha stimulated [3H]thymidine incorporation and tubulogenesis of RGM1 cells in collagen matrix, both of which were significantly suppressed by treatment with a cyclooxygenase-2 specific inhibitor, NS-398 or cyclooxygenase-2 antisense oligonucleotide. Both of the treatment lowered prostanoid production by enzyme immunoassay. The transforming growth factor alpha-induced expression of cyclooxygenase-2 is followed by cell proliferation and development of tubular morphology of RGM1 gastric epithelial cells. Treatment with cyclooxygenase-2 inhibitor and cyclooxygenase-2 antisense oligonucleotide suppressed these responses induced by transforming growth factor alpha suggesting the involvement of cyclooxygenase-2 in proliferation and morphogenesis in gastric mucosal epithelium.     

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-α and epidermal growth factor in vivo

Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-α and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-α or EGF has a functional effect on the olfactory mucosa in vivo, 12.5–50 μg of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-α activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-α was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-α to EGFR may play a role in olfactory cell division in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 199–210, 1998     

Anti-(epidermal growth factor) receptor monoclonal antibodies for the induction of antibody-dependent cell-mediated cytotoxicity against squamous cell carcinoma lines of the head and neck

 Squamous cell carcinomas of the head and neck (SCCHN) frequently display high levels of the epidermal growth factor receptor (EGFR). Since EGFR is expressed on the cell surface it may form a suitable target for anticancer therapy with anti-receptor monoclonal antibodies (mAb). Besides the interference with receptor/ligand interactions, binding of mAb to EGFR leads to immunoglobulin-coated tumour cells that may induce or enhance non-specific immune effector mechanisms like antibody-dependent cell-mediated cytotoxicity (ADCC). In established cell lines of SCCHN (UM-SCC 11B, 14C, 22B, and 8029 NA) we investigated the antitumour activity of allogeneic peripheral blood mononuclear cells (PBMC) in combination with rat (ICR 62), mouse (EMD 55900), and humanized (EMD 72000) anti-EGFR mAb. In addition, autologous PBMC were available for tumour line UD-SCC 4. The EGFR protein content of the tumour cell lines ranged between 170 fmol/mg protein and 8100 fmol/mg protein, and MCF-7 cells served as receptor-negative controls. PBMC activity against SCCHN targets was determined in 96-well microtitre-plate monolayer cultures by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after coincubation for 4 h, 24 h and 72 h at effector target ratios of 1:1, 5:1, 10:1 and 20:1. PBMC subpopulations were obtained by macrophage depletion (plastic adherence) or natural killer (NK) cell enrichment (magnetic bead negative selection). Prolonged time of exposure and increased effector:target ratios revealed marked antitumour activity of PBMC alone. This non-specific immune destruction was enhanced considerably by humanized and rat, but not mouse anti-EGFR mAb. Increased EGFR protein in tumour cells partly correlated with an intensification of ADCC but was accompanied by decreased primary PBMC cytotoxicity. The utilization of PBMC subpopulations suggested a mainly NK-cell-mediated ADCC, which appeared to benefit directly or indirectly, e.g. via the secretion of cytokines, from other PBMC components. In conclusion, humanized (EMD 72000) and rat (ICR 62) anti-EGFR mAb were able to generate strong antitumour ADCC in target monolayers of SCCHN. Received: 5 December 1997 / Accepted: 15 January 1998     

Transforming growth factor-β1 is an autocrine mediator of U937 cell growth arrest and differentiation induced by vitamin D3 and retinoids

Vitamin D and retinoids cooperate to inhibit the proliferation and induce the differentiation of human myelomonocytic U937 leukemia cells. In the present work, we investigated the role of TGF-β as an endogenous mediator of this process. We found that the TGF-β1 precursor began to accumulate in cell culture supernatants soon after the addition of 1α,25 dihydroxyvitamin D₃ (VD) and retinoids. We used neutralizing antibodies (AbTGF-β) and antisense oligonucleotide (AS Oligo) to inhibit its possible effects. Our data demonstrated that AbTGF-β partially inhibit the expression of the differentiated phenotype, as assessed by measurement of phagocytic activity, response to the chemotactic peptide fMLP, and lysozyme secretion. AS Oligo was also inhibitory, and the effects of AS Oligo and AbTGF-β were cumulative. Cell growth inhibition induced by VD and retinoids was completely reversed, and differentiation was reduced by about 75% when both inhibitors were associated. Time course experiments based on the delayed addition of AbTGF-β and AS Oligo showed that TGF-β1 was required for cell differentiation 24 h after the addition of inducers. Studies on TGF-β receptors revealed that, while the expression of type II receptor was stable, the level of type I TGF-β receptor mRNA and the expression of the protein began to decline early during the differentiation process. As a whole, these results support the notion that an autocrine TGF-β pathway, activated by VD and retinoids in U937 cells, is involved in the early steps of the process leading to cell growth arrest and differentiation. J Cell Physiol 178:109–119, 1999. © 1999 Wiley-Liss, Inc.     

The tetraspanin CD9 associates with transmembrane TGF-alpha and regulates TGF-alpha-induced EGF receptor activation and cell proliferation

Transforming growth factor-alpha (TGF-alpha) is a member of the EGF growth factor family. Both transmembrane TGF-alpha and the proteolytically released soluble TGF-alpha can bind to the EGF/TGF-alpha tyrosine kinase receptor (EGFR) and activate the EGFR-induced signaling pathways. We now demonstrate that transmembrane TGF-alpha physically interacts with CD9, a protein with four membrane spanning domains that is frequently coexpressed with TGF-alpha in carcinomas. This interaction was mediated through the extracellular domain of transmembrane TGF-alpha. CD9 expression strongly decreased the growth factor- and PMA- induced proteolytic conversions of transmembrane to soluble TGF-alpha and strongly enhanced the TGF- alpha-induced EGFR activation, presumably in conjunction with increased expression of transmembrane TGF-alpha. In juxtacrine assays, the CD9-induced EGFR hyperactivation by transmembrane TGF-alpha resulted in increased proliferation. In contrast, CD9 coexpression with transmembrane TGF-alpha decreased the autocrine growth stimulatory effect of TGF-alpha in epithelial cells. This decrease was associated with increased expression of the cdk inhibitor, p21(CIP1). These data reveal that the association of CD9 with transmembrane TGF-alpha regulates ligand-induced activation of the EGFR, and results in altered cell proliferation.     

Autocrine and juxtacrine effects of amphiregulin on the proliferative, invasive, and migratory properties of normal and neoplastic human mammary epithelial cells

Amphiregulin (AR) autocrine loops have been associated with several types of cancer. We demonstrate that SUM149 breast cancer cells have a self-sustaining AR autocrine loop. SUM149 cells are epidermal growth factor (EGF)-independent for growth, and they overexpress AR mRNA, AR membrane precursor protein, and secreted AR relative to the EGF-dependent human mammary epithelial cell line MCF10A. MCF10A cells made to overexpress AR (MCF10A AR) are also EGF-independent for growth. Treatment with the pan-ErbB inhibitor CI1033 and the anti-EGF receptor (EGFR) antibody C225 demonstrated that ligand-mediated activation of EGFR is required for SUM149 cell proliferation. AR-neutralizing antibody significantly reduced both SUM149 EGFR activity and cell proliferation, confirming that an AR autocrine loop is required for mitogenesis in SUM149 cells. EGFR tyrosine phosphorylation was dramatically decreased in both SUM149 and MCF10A AR cells after inhibition of AR cleavage with the broad spectrum metalloprotease inhibitor GM6001, indicating that an AR autocrine loop is strictly dependent on AR cleavage in culture. However, a juxtacrine assay where fixed SUM149 cells and MCF10A AR cells were overlaid on top of EGF-deprived MCF10A cells showed that the AR membrane precursor can activate EGFR. SUM149 cells, MCF10A AR cells, and MCF10A cells growing in exogenous AR were all considerably more invasive and motile than MCF10A cells grown in EGF. Moreover, AR up-regulates a number of genes involved in cell motility and invasion in MCF10A cells, suggesting that an AR autocrine loop contributes to the aggressive breast cancer phenotype.     

Nimotuzumab,a promising therapeutic monoclonal for treatment of tumors of epithelial origin

Nimotuzumab is a humanized therapeutic monoclonal antibody against epidermal growth factor receptor (EGFR). Clinical trials are ongoing globally to evaluate nimotuzumab in different indications. Nimotuzumab has been granted approval for use in squamous cell carcinoma of head and neck (SCCHN), glioma and nasopharyngeal cancer in different countries. This review focuses on the unique functional characteristics of nimotuzumab. Also, it discusses the safety and efficacy data obtained from the Phase IIb clinical trial conducted in India in SCCHN. Post Marketing Surveillance data from Cuba for the use of nimotuzumab in pediatric and adult glioma is also discussed. Overall, nimotuzumab has immense therapeutic potential in cancers of epithelial origin.     

Critical role of both p27KIP1 and p21CIP1/WAF1 in the antiproliferative effect of ZD1839 ('Iressa'), an epidermal growth factor receptor tyrosine kinase inhibitor,in head and neck squamous carcinoma cells

High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of squamous-cell carcinomas of head and neck (SCCHN). ZD1839 ('Iressa') is an orally active, selective EGFR-TKI (EGFR-tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. We have demonstrated that ZD1839 induces growth arrest in SCCHN cell lines by inhibiting EGFR-mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in cell cycle progression and a G1 arrest together with a partial G2/M block; this was associated with increased expression of both p27(KIP1) and p21(CIP1/WAF1) cyclin-dependent kinase (CDK) inhibitors. The activity of CDK2, the main target of CIP/KIP CDK inhibitors, was reduced in a dose-dependent fashion after 24 h of ZD1839 treatment and this effect correlated to the increased amount of p27(KIP1) and p21(CIP1/WAF1) proteins associated with CDK2-cyclin-E and CDK2-cyclin-A complexes. In addition, ZD1839-induced growth inhibition was significantly reduced in cell transfectants expressing p27(KIP1) or p21(CIP1/WAF1) antisense constructs. Overall, these results as well as the timing of the effect of ZD1839 on G1 arrest and p27(KIP1) and p21(CIP1/WAF1) upregulation, suggest a mechanistic connection between these events.