RESUMO
PURPOSE: Activation of the epidermal growth factor receptor (EGFR) requires cell surface cleavage of EGFR ligands, uptake of soluble ligand by the receptor, and initiation of EGFR tyrosine kinase activity. We define these collective events as the EGFR axis. Transforming growth factor-alpha (TGF-alpha) and amphiregulin are two EGFR ligands that are delivered preferentially to the basolateral surface of polarized epithelial cells where the EGFR resides. TACE/ADAM-17 (tumor necrosis factor-alpha converting enzyme/a disintegrin and metalloprotease) has been implicated in ectodomain cleavage of TGF-alpha and amphiregulin. EXPERIMENTAL DESIGN: Using a human polarizing colorectal cancer (CRC) cell line, HCA-7, and a tissue array of normal colonic mucosa and primary and metastatic CRC, we determined the intracellular localization of TACE and the effects of EGFR axis inhibition in CRC. RESULTS: Herein, we show that TACE is localized to the basolateral plasma membrane of polarized HCA-7 cells. TACE is overexpressed in primary and metastatic CRC tumors compared with normal colonic mucosa; the intensity of its immunoreactivity is inversely correlated with that of TGF-alpha and amphiregulin. Pharmacologic blockade of HCA-7 cells with an EGFR monoclonal antibody, a selective EGFR tyrosine kinase inhibitor, and a selective TACE inhibitor results in concentration-dependent decreases in cell proliferation and active, phosphorylated mitogen-activated protein kinase. Combining suboptimal concentrations of these agents results in cooperative growth inhibition, increased apoptosis, and reduced mitogen-activated protein kinase pathway activation. Furthermore, an EGFR tyrosine kinase-resistant clone of HCA-7 cells is growth-inhibited by combined monoclonal antibody and TACE inhibition. CONCLUSION: These results implicate TACE as a promising target of EGFR axis inhibition in CRC.
Assuntos
Proteínas ADAM/metabolismo , Neoplasias Colorretais/enzimologia , Receptores ErbB/metabolismo , Proteína ADAM17 , Anfirregulina , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Família de Proteínas EGF , Inibidores Enzimáticos/farmacologia , Receptores ErbB/efeitos dos fármacos , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Análise Serial de Tecidos , Fator de Crescimento Transformador alfa/metabolismoRESUMO
PURPOSE: To evaluate noninvasive molecular imaging methods as correlative biomarkers of therapeutic efficacy of cetuximab in human colorectal cancer cell line xenografts grown in athymic nude mice. The correlation between molecular imaging and immunohistochemical analysis to quantify epidermal growth factor (EGF) binding, apoptosis, and proliferation was evaluated in treated and untreated tumor-bearing cohorts. EXPERIMENTAL DESIGN: Optical imaging probes targeting EGF receptor (EGFR) expression (NIR800-EGF) and apoptosis (NIR700-Annexin V) were synthesized and evaluated in vitro and in vivo. Proliferation was assessed by 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) positron emission tomography. Assessment of inhibition of EGFR signaling by cetuximab was accomplished by concomitant imaging of NIR800-EGF, NIR700-Annexin V, and [18F]FLT in cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell line xenografts. Imaging results were validated by measurement of tumor size and immunohistochemical analysis of total and phosphorylated EGFR, caspase-3, and Ki-67 immediately following in vivo imaging. RESULTS: NIR800-EGF accumulation in tumors reflected relative EGFR expression and EGFR occupancy by cetuximab. NIR700-Annexin V accumulation correlated with cetuximab-induced apoptosis as assessed by immunohistochemical staining of caspase-3. No significant difference in tumor proliferation was noted between treated and untreated animals by [18F]FLT positron emission tomography or Ki-67 immunohistochemistry. CONCLUSIONS: Molecular imaging can accurately assess EGF binding, proliferation, and apoptosis in human colorectal cancer xenografts. These imaging approaches may prove useful for serial, noninvasive monitoring of the biological effects of EGFR inhibition in preclinical studies. It is anticipated that these assays can be adapted for clinical use.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Diagnóstico por Imagem/métodos , Receptores ErbB/metabolismo , Animais , Anticorpos Monoclonais Humanizados , Cetuximab , Neoplasias Colorretais/diagnóstico por imagem , Receptores ErbB/efeitos dos fármacos , Radioisótopos de Flúor , Humanos , Interpretação de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Espectroscopia de Luz Próxima ao Infravermelho , Timidina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The peripheral benzodiazepine receptor (PBR) is a trans-mitochondrial membrane protein that modulates steroid biosynthesis. Recently, up-regulation and nuclear localization of PBR has been shown to be associated with colon, prostate, and breast cancer. PBR has been targeted by the exogenous synthetic ligand, PK11195, for various purposes including imaging. To capitalize on these observations, we developed a high-throughput, noninvasive, in vivo imaging approach to detect spontaneously arising colonic tumors in mice using a novel PBR-targeted molecular imaging agent (NIR-conPK11195). NIR-conPK11195 localized and was retained in colonic adenomas and carcinomas in Smad3(-/-) mice but not in non-neoplastic hamartomas or chronically inflamed colonic tissue. Using a fluorescence signal-to-noise ratio of > or =4-fold 13 h after injection of the agent, we detected colonic tumors with a sensitivity of 67% and a specificity of 86% in a cohort of 37 Smad3(-/-) mice and control littermates. Furthermore, using oral administration of dextran sulfate to induce colonic inflammation, we showed that the clearance profile of NIR-conPK11195 distinguished transient uptake in inflammatory tissue from longer term retention in tumors. Taken together, these results indicate that NIR-conPK11195 is a promising optical molecular imaging tool to rapidly screen for colonic tumors in mice and to discriminate inflammation from cancer.