SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae function.

Caveolae are a major membrane domain common to most cells. One of the defining features of this domain is the protein caveolin. The exact function of caveolin, however, is not clear. One possible function is to attract adapter molecules to caveolae in a manner similar to how clathrin attracts molecules to coated pits. Here, we characterize a candidate adapter molecule called SRBC. SRBC binds PKCdelta and is a member of the STICK (substrates that interact with C-kinase) superfamily of PKC-binding proteins. We also show it co-immunoprecipitates with caveolin-1. A leucine zipper in SRBC is essential for both co-precipitation with caveolin and localization to caveolae. SRBC remains associated with caveolin when caveolae bud to form vesicles (cavicles) that travel on microtubules to different regions of the cell. In the absence of SRBC, intracellular cavicle traffic is markedly impaired. We conclude that SRBC (sdr-related gene product that binds to c-kinase) and two other family members [PTRF (Pol I and transcription release factor) and SDPR] function as caveolin adapter molecules that regulate caveolae function.

Direct sequencing in cytological specimens as a useful strategy for detecting EGFR mutations in non-small cell lung cancer patients.

BACKGROUND: New therapeutics targeting epidermal growth factor receptor (EGFR) have significantly improved tumor responses to therapy in non-small cell lung cancer (NSCLC) patients. Molecular testing for EGFR mutations informs important therapeutic decisions in clinical practice. In this study, we sought to validate the clinical relevance of sequencing-based EGFR mutation testing combined with cytological analysis using body fluid specimens. METHODS: Two NSCLC cell lines were used in sensitivity analyses. In addition, we performed cytological analyses and directly sequencing of exons 18-21, for 32 specimens. The absence of EGFR mutations determined by direct sequencing in 14 specimens was confirmed by real-time PCR. Changes made to patients' therapeutic strategies after reports of EGFR mutation status were investigated by querying electronic medical records. RESULTS: Sensitivity studies showed that detection of in-frame deletions in exon 19 and point mutations in exon 21 was possible in specimens containing 10% and 5% mutant DNA, respectively. In clinical practice, EGFR mutations were detected in 18 of 32 specimens (56.3%). Twelve patients with EGFR mutations detected by direct sequencing were started on treatment with EGFR tyrosine kinase inhibitor (TKI) after reports of EGFR mutation. EGFR-TKI therapy was discontinued for two patients with TKI-resistant T790M mutation. The results of real-time PCR were consistent with those of direct sequencing in 13 of 14 specimens (92.9%) in which no mutation was detected by direct sequencing. CONCLUSIONS: Combined direct sequencing and cytological analysis of body fluid specimen might be clinically useful and sensitive test for the detection of EGFR mutations in NSCLC patients.

Implications of EGFR inhibition in ovarian cancer cell proliferation.

OBJECTIVES: Epidermal Growth Factor Receptor (EGFR) is one of the four members of the Human Epidermal Receptor (HER) family and is over-expressed in multiple malignancies. EGFR over-expression in ovarian cancer has been associated with poor prognosis. Targeted inhibition of EGFR via its tyrosine kinase domain is a successful treatment in lung cancer. Our objective was to correlate EGFR over-expression and growth inhibition, by EGF receptor inhibitors, in ovarian cancer. MATERIALS AND METHODS: HER expression in nine epithelial ovarian cancer cell lines and one lung cancer cell line was determined by Western blot analysis. EGFR phosphorylation sites were analyzed and DNA sequencing was performed. Cell proliferation assays were performed in the presence of the tyrosine kinase inhibitor, gefitinib, and the EGFR monoclonal antibody, cetuximab. Inhibitory concentrations of 50% of these therapies were determined and compared across all cell lines. The lung cancer cell line, HCC827, was used as a control. RESULTS: Four of nine (44%) ovarian cancer cell lines and the control lung cancer cell line expressed EGFR. These same cell lines showed a common phosphorylated residue at position 992, while other residues were variably phosphorylated. All but one cell line expressed at least one HER family member. Mutational analysis of the ovarian cancer cell lines showed no mutations in EGFR exons 18-21. Cell proliferation assays using gefitinib and cetuximab showed minimal response in the ovarian cancer cell lines when compared to the control HCC827, but relative sensitivity compared to the one cell line that had no HER family expression. CONCLUSIONS: Ovarian cancer cell lines show variable expression of activated EGFR. EGFR inhibition alone, in ovarian tumors that lack a tyrosine kinase mutation or over-express EGFR is unlikely to result in clinical response.