Lung Adenocarcinoma and Its Stroma: Tissue Proteomics Reveals Differential and Compartment-Specific Roles for the Homologs Transgelin and Transgelin-2
MH Roehrl, JH Rho, JY Wang. Massachusetts General Hospital, Boston, MA; Brigham and Women's Hospital, Boston, MA
Background: Early detection of lung adenocarcinoma progression and discovery of underlying functional mechanisms are hampered by a large dynamic concentration range of tissue biomacromolecules and the fact that current proteomic approaches are limited by the presence of relatively few highly abundant proteins.
Design: To overcome this limitation, we first developed heparin affinity chromatography enrichment (HAFE) as a powerful technique to significantly enrich for low-abundance components. HAFE-enriched protein extracts from five fresh-frozen paired normal and adenocarcinoma tissues were then studied by 2-D difference gel electrophoresis (DIGE) and tandem mass spectrometry (MS/MS). Additional independent paired tissue samples were studied by Western blotting, immunohistochemistry, and RT-PCR.
Results: Fourteen proteins were found differentially expressed in neoplastic versus normal tissue. The identities of three protein spots were elucidated by tandem mass spectrometric (MS/MS) sequencing as transgelin (TAGLN), transgelin-2 (TAGLN2), and cyclophilin A (PPIA). All three proteins were overexpressed in lung adenocarcinoma tissue. The protein expression levels were further validated by Western blotting of both unfractionated total protein and HAFE-enriched extracts from 15 paired lung adenocarcinoma samples. Four paired lung squamous cell carcinoma and 9 paired colon adenocarcinoma samples were also studied as controls. Semi-quantitative RT-PCR indicated that both TAGLN2 and PPIA are upregulated at transcriptional level. Intriguingly, we found by immunohistochemistry that the overexpression of TAGLN was strictly localized to the tumor-induced reactive stromal tissue compartment, while the overexpression of TAGLN2 was exclusively localized to the neoplastic glandular compartment.
Conclusions: We discovered that two homologous proteins, TAGLN and TAGLN2, which possess 87% amino acid sequence similarity, display striking mutually exclusive and complementary spatial and cell type expression regulation (i.e., tumor stroma vs. neoplastic epithelial cells). Our work describes the discovery of novel candidate biomarkers of lung adenocarcinoma that may advance basic pathophysiologic understanding of lung adenocarcinoma, as well as early diagnosis, treatment guidance, and treatment response monitoring.
Category: Special Category for 2009 - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, March 9, 2009 9:30 AM
Poster Session I Stowell-Orbison/Autopsy Award # 242, Monday Morning