Maihle, Nita J.

Abstract A circulating isoform of EGFR has been identified as an emerging serum biomarker in cancer patients. Serum concentrations of EGFR have been evaluated as screening, diagnostic, prognostic, and theragnostic markers for diverse human malignancies, including breast, ovarian, endometrial, colorectal, and lung cancer. Yet full-length EGFR is rarely shed from normal or malignant cells. Here we demonstrate that an alternately spliced EGFR isoform, termed ‘sEGFR,’ gives rise to the circulating sEGFR protein through proteolytic cleavage of a cell surface precursor. This cleavage event is inhibited by fibronectin and stimulated by both an anti-Δ5/α1 integrin antibody and the metalloprotease activator 4-aminophenylmercuric acetate. Preliminary results identify ADAM17/TACE as at least one mediator of sEGFR shedding. Shed sEGFR binds to EGF with high affinity. Two FDA-approved therapeutic anti-EGFR antibodies, cetuximab (Erbitux) and panitumumab (Vectibix), inhibit shedding of sEGFR, implicating sEGFR as a potential alternative target for these antibodies, consistent with previous studies indicating that baseline serum sEGFR concentrations may predict responsiveness to anti-EGFR-targeted therapy. These observations bear striking similarity to aspects of trastuzumab regulation of HER2, shedding, and, by analogy, may have implications for the design of future clinical trials using EGFR-directed therapeutics, including cetuximab and panitumimab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3624. doi:1538-7445.AM2012-3624

Abstract In July, 2009 the United States’ Food and Drug Agency (FDA) approved the use of a new biologically-targeted drug called ‘cetuximab’ (Erbitux) for the treatment of patients with advanced colorectal cancer (CRC). Cetuximab is a chimeric antibody directed against the extracellular domain of the epidermal growth factor receptor (EGFR). Paradoxically, EGFR expression, as it is currently measured by commercially available assays, has not been useful for predicting who will respond to cetuximab. We propose that one problem with these assays is their inability to measure EGFR and soluble EGFR (sEGFR) isoforms independently. We previously have documented the expression of an sEGFR isoform in human tissues (1), and have generated antibodies directed against a 31-mer peptide (residues 604-634) located within the unique 78 amino acid carboxy-terminal sequence of sEGFR (2). Interestingly, this anti-sEGFR antibody recognizes a second protein of ∼140-kDa (p140), which we have identified as human α5-integrin. Biochemical and mass spectrometry analyses show that a short region in the calf domain of α5-integrin shares sequence identity with the 31-mer sEGFR peptide. We have used this 31-mer antibody with an antibody directed against the intracellular domain of full-length EGFR to develop a novel EGFR vs. sEGFR/α5-integrin expression assay. This assay demonstrates that sEGFR/α5-integrin is co-expressed with EGFR in normal as well as malignant tissues, including in a number of carcinoma-derived cell lines. We also observe that high expression of EGFR and sEGFR/α5-integrin in a large cohort of ovarian cancer patients defines a strong adverse prognostic determinant of survival. These results implicate EGFR, sEGFR, and integrins as cancer biomarkers and demonstrate the critical importance of independently assessing both full-length and soluble EGFR isoforms in prognostic EGFR expression assays. *–JAW and ATB contributed equally to this study (1). Reiter et al. (2001) Genomics. 71(1):1-20 (2). Christensen et al. (2002) Hybrid Hybridomics. 21(3):183-9 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 4649.

After therapeutic hormone deprivation, prostate cancer cells often develop androgen-insensitive growth through mechanisms thus far undefined. Neuropeptides have been previously implicated as growth factors in some prostate cancers. Here, we demonstrate that androgen-sensitive LNCaP human prostate cancer cells produce and secrete neurotensin following androgen withdrawal. We show that while LNCaP cells express the neurotensin receptor, only androgen-deprived cells exhibit a growth response to exogenous neurotensin. We further demonstrate that androgen-stimulated cells may be refractory to exogenous neurotensin due to androgen induction of a metalloprotease active toward neurotensin. Thus, prostate cancer cells deprived of androgen develop an alternative autocrine growth mechanism involving neurotensin.

A primer-directed cDNA library was used to obtain cDNA clones corresponding to the 5' end (i.e., the ligand-binding domain) of the avian c-erbB gene. Bacterial c-erbB fusion proteins were synthesized and used to obtain polyclonal antisera specific for the ligand-binding domain of the avian receptor. These antisera and antisera specific for the carboxyl terminal domain of the chicken c-erbB gene product have been used to study the native protein products of the c-erbB locus in primary cell cultures by in vivo labeling and immunoprecipitation. Our studies reveal that three c-erbB gene products of Mr 300,000, Mr 170,000, and Mr 95,000 are synthesized in uninfected chicken embryo fibroblasts. Only the Mr 300,000 and Mr 170,000 species can be precipitated by using antisera specific for the cytoplasmic domain of the c-erbB product. The 95,000 species is not recognized by the antiserum directed against the carboxyl-terminal domain of c-erbB and is specifically released into the culture medium. Northern transfer studies reveal a lower molecular weight transcript of approximately 2.6 kilobases that selectively hybridizes to the ligand-binding domain of the avian c-erbB gene product but does not hybridize with probes specific for the cytoplasmic kinase domain of c-erbB. An additional cDNA clone corresponding to this transcript has been isolated, and its sequence suggests it may arise via alternative processing. Together, these data suggest that a truncated form of this growth factor receptor--i.e., a Mr 95,000 species--is synthesized from a low molecular weight c-erbB transcript that exclusively encodes the ligand-binding domain of the receptor. Secretion of truncated growth factor receptors has been reported recently in several systems, and our results are discussed in the light of these findings.

Abstract Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecologic cancers in the United States. Because women who are diagnosed with early stage disease have a better prognosis than women diagnosed with late stage disease, early detection represents a potentially practical approach to reduce the mortality associated with EOC. Unfortunately, no single screening test has proven to be effective for this purpose, and a valid and feasible screening program to detect early stage EOC in the general population has not yet been devised. Consequently, research has focused on coupling two or more screening modalities to improve program validity and feasibility. Serum cancer antigen 125 (CA125) and a soluble isoform of the epidermal growth factor receptor (p110 sEGFR) have been studied individually as biomarkers of ovarian cancer. In this study, we compare serum CA125 levels and sEGFR concentrations in women with EOC to women with benign gynecologic conditions of ovarian and non-ovarian origin. We show that serum sEGFR concentrations are lower in patients with EOC than in women with benign gynecologic conditions, whereas serum CA125 levels are higher in patients to EOC compared with women with benign gynecologic conditions. These data also reveal that age and serum sEGFR concentrations modify the association between CA125 levels and EOC versus benign gynecologic disease. Hence, age- and sEGFR-dependent CA125 cutoff thresholds improve the ability of CA125 to discern EOC patients from women with benign ovarian tumors and non-ovarian gynecologic conditions. Our analyses show that parallel testing with fixed sEGFR and CA125 cutoff thresholds optimizes sensitivity to detect EOC, whereas serial testing with age- and sEGFR-dependent CA125 cutoff thresholds optimizes test specificity, and overall accuracy to discern patients with EOC from women with benign ovarian and non-ovarian gynecologic conditions. The combined use of serologic sEGFR and CA125, thus, has improved utility for screening and diagnosing EOC, which may increase the positive predictive value of a multimodal screening program that incorporates these biomarkers to detect and subsequently differentiate benign from malignant ovarian tumors.

The centrosome plays an important role in maintenance of cell polarity and in progression through the cell cycle by determining the number, polarity, and organization of interphase and mitotic microtubules. By examining a set of 35 high grade human breast tumors, we show that centrosomes of adenocarcinoma cells generally display abnormal structure, aberrant protein phosphorylation, and increased microtubule nucleating capacity in comparison to centrosomes of normal breast epithelial and stromal tissues. These structural and functional centrosome defects have important implications for understanding the mechanisms by which genomic instability and loss of cell polarity develop in solid tumors.

The avian erythroblastosis viral oncogene (v- erb B) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v- erb B in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v- erb B-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc–Grb2–Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.