Expression of EGFR isoform D is regulated by HER receptor activators in breast cancer cells.

Human epidermal growth factor receptor isoform D (EGFR; isoform D) is a soluble protein from a 3 kb alternate mRNA transcript that arises from the human EGFR gene. Several studies have identified this circulating isoform of EGFR as a potential diagnostic biomarker for the detection of early stage of cancers. While the expression of the full-length EGFR (isoform A) is regulated by its cognate ligand, EGF, as well as by phorbol myristate acetate (PMA), no studies have examined the factors regulating the expression of EGFR isoform D. In this study, using breast cancer cell lines, we show that the HER receptor ligands, EGF and neuregulin (NRG-1ß), as well as the phorbol ester, PMA, can increase the expression of EGFR isoform D, as well as isoform A. Our results, based on measurement of mRNA levels, suggest that EGF induced expression of both isoform A and isoform D occur through a mitogen activated protein kinase (MAPK)-dependent mechanism, and also suggest that protein kinase C is involved in PMA-induced regulation of both isoforms. We also demonstrate that NRG-1ß increases isoform A and isoform D expression via the MAPK-dependent pathway, but this regulation occurs independently of phosphatidylinositol 3-kinase/Akt activation. These results suggest that regulation of EGFR isoform A and isoform D expression occur using similar mechanisms. Despite commonalities in the transcriptional regulation of these two EGFR isoforms, the half-lives of these two transcripts is quite different. Moreover, EGFR isoform D, unlike isoform A, is not post-transcriptionally modulated by EGFR activators in the breast cancer cell line MDA-MB-468.

Shedding of soluble epidermal growth factor receptor (sEGFR) is mediated by a metalloprotease/fibronectin/integrin axis and inhibited by cetuximab.

Soluble epidermal growth factor receptor (sEGFR) is a circulating serum biomarker in cancer patients. Recent studies suggest that baseline serum sEGFR concentrations may predict responsiveness to EGFR-targeted therapy. Here, we demonstrate that sEGFR is generated through proteolytic cleavage of a cell surface precursor of an alternately spliced EGF receptor isoform and that sEGFR binds to EGF with high affinity. Proteolytic cleavage is stimulated by an anti-α5/ß1 integrin antibody and 4-aminophenylmercuric acetate, and inhibited by fibronectin. Two FDA-approved therapeutic anti-EGFR antibodies also inhibit shedding of sEGFR, thus implicating the cell surface precursor of sEGFR as a competing target for anti-EGFR antibodies in human tissues. These observations parallel trastuzumab regulation of HER2 shedding and have implications for patient stratification in future clinical trials of EGFR-targeted antibodies.

Phase II, randomized trial to compare anastrozole combined with gefitinib or placebo in postmenopausal women with hormone receptor-positive metastatic breast cancer.

PURPOSE: This phase II randomized trial evaluated the efficacy and tolerability of anastrozole combined with gefitinib or anastrozole with placebo in women with hormone receptor-positive metastatic breast cancer (MBC). EXPERIMENTAL DESIGN: Postmenopausal women with hormone receptor-positive measurable or evaluable MBC who had not received prior endocrine therapy for this disease stage or who developed metastatic disease during/after adjuvant tamoxifen were eligible. The primary response variable was progression-free survival (PFS) and secondary response variables included clinical benefit rate, objective response rate, overall survival, safety and tolerability, and pharmacokinetics. Tumor biomarker evaluation was an exploratory objective. RESULTS: Forty-three patients were randomized to anastrozole plus gefitinib and 50 patients were randomized to anastrozole plus placebo of a planned total of 174 patients (enrollment was prematurely discontinued due to slow recruitment). PFS for patients receiving the combination of anastrozole and gefitinib was longer than for patients receiving anastrozole plus placebo [hazard ratio (gefitinib/placebo), 0.55; 95% confidence interval, 0.32-0.94; median PFS, 14.7 versus 8.4 months]. The clinical benefit rate was 49% versus 34%, and the objective response rate was 2% versus 12% with anastrozole plus gefitinib and anastrozole plus placebo, respectively. No evidence of interaction between baseline biomarker levels and relative treatment effect was found. No unexpected adverse events were observed. CONCLUSION: This small randomized study showed that anastrozole in combination with gefitinib is associated with a marked advantage in PFS compared with anastrozole plus placebo, and that the combination was tolerated in postmenopausal women with hormone receptor-positive MBC. Further investigation of epidermal growth factor receptor inhibition in combination with endocrine therapy may be warranted.

Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells.

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.

Serum soluble epidermal growth factor receptor concentrations decrease in postmenopausal metastatic breast cancer patients treated with letrozole.

Previous studies have implicated estrogen as a regulator of epidermal growth factor receptor (EGFR) expression in breast tumors. We therefore speculated that estrogen might modulate serologic soluble EGFR (sEGFR) concentrations in breast cancer patients. Accordingly, we measured serum sEGFR concentrations in postmenopausal women with metastatic breast cancer (MBC) treated with letrozole, an aromatase inhibitor that blocks estrogen synthesis. Serum specimens were obtained prior to and following 1 and 3 months of letrozole therapy. We report that sEGFR concentrations do not differ between MBC patients prior to letrozole treatment and age- and postmenopause-matched healthy women (P = 0.468). In contrast, however, sEGFR concentrations decreased significantly in 76% of MBC patients after both 1 month (P = 0.006) and 3 months (P = 0.003) of letrozole therapy versus pretreatment concentrations. Within the limitations of this study, we found no evidence for an association between pretreatment sEGFR concentrations or decreased treatment sEGFR concentrations and either progression-free or overall survival. Nonetheless, we conclude that future prospective studies are warranted to determine if baseline and/or longitudinal serum sEGFR concentrations may be useful for predicting disease progression and survival, and/or for monitoring responsiveness to aromatase inhibitors or other endocrine therapies in breast cancer patients.

Soluble epidermal growth factor receptor (sEGFR/sErbB1) as a potential risk, screening, and diagnostic serum biomarker of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies in the United States, for which risk assessment, screening, and diagnostic tests are needed. We have shown previously that women with stage III/IV EOC have lower serum p110 sEGFR/sErbB1 (Soluble Epidermal Growth Factor Receptor) concentrations than healthy women. Here, we show that serum p110 sEGFR/sErbB1 is the product of a 3-kb EGFR/ERBB1 alternate transcript. We report that serum sEGFR concentrations in stage I/II and stage III/IV EOC patients are significantly lower than in healthy women, and that serum sEGFR concentrations are not associated with disease stage or tumor grade. Logistic regression models show that: (a) lower serum sEGFR concentrations are associated significantly with a greater risk of EOC; (b) the risk associated with lower serum sEGFR concentrations is reduced by older age or menopause; and (c) age- or menopausal status-specific cutoff values for sEGFR concentration are appropriate. Receiver operating characteristic curves indicate that: (a) serum sEGFR concentrations are more effective in discerning stage III/IV than stage I/II EOC cases from healthy women; and (b) sEGFR concentrations have an 89% probability of correctly discerning EOC patients from healthy women when accounting for effect modification by age. By maintaining a test specificity of approximately 95% across strata of age or menopausal status with appropriate cutoff values, we observe that sEGFR concentrations are most useful for detecting stage I/II (sensitivity: 64-67%) and stage III/IV (sensitivity: 75-81%) EOC in young, premenopausal women. We conclude that serum sEGFR concentrations warrant additional investigation in the risk assessment, early detection, and/or diagnosis of EOC.