Proteomic analysis of beta-catenin during the neoplastic progression from precursor lesions to invasive squamous cell carcinoma of the esophagus

MJ Roth, MD,a N Hu, MD,a CP Paweletz, MS,a M Iwamoto, MD,a Y-L Qiao, MD,b W-J Li, MD,c H. Su, PhD,a DJ Ahnen, MD,d SM Dawsey, MD,a PR Taylor, MD,a

aNational Cancer Institute, Bethesda, MD bCancer Institute, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China, cShanxi Cancer Hospital and Institute, Taiyuan, Shanxi, People’s Republic of China, dUniversity of Colorado Health Sciences Center, Denver, CO

AIM: To better understand the proteomic and molecular alterations of beta-catenin (3p22-p21.3) associated with the neoplastic progression of squamous esophageal cancer (EC). METHODS: Pathologic specimens from the high-risk areas of Linxian and Shanxi were analyzed. Laser Capture Microdissection was used to procure pure populations of cells for all molecular and proteomic assays. Immunohistochemistry: performed on normal (NL), low-grade dysplasia (LG), high-grade dysplasia (HG) and invasive cancer (CA) foci in 7 esophagectomies (32 foci total). LOH and Mutational Analysis: performed on 56 cases using 5 polymorphic markers surrounding beta-catenin (D3S3527, D3SW3442, MR01 (WIAF-884), D3S3152, and D3S2417) and SSCP DNA sequencing gels. Protein Microarray: Protein lysates were prepared of NL, LG, HG and CA foci in 8 cases (36 foci total). RESULTS: Immunohistochemistry: Microscopy identified an increase in beta-catenin expression in precancerous and cancerous lesions versus NL foci. Expression in dysplastic epithelium was greater than in invasive cancer. LOH and Mutational Analysis: 3/5 LOH markers were uninformative (homozygous) for all 56 cases. 2/5 markers showed 64% (D3S3527) and 100% (D3S2417) LOH. Two unique mutations were identified in two of the 56 cases, consisting of a somatic tct---ttt change (S37F), resulting in a serine to phenylalanine substitution in the SxxxS repeat region, and a germline polymorphism gcc---acc change (T59A) resulting in an alanine to threonine substitution. Protein Microarray: Preinvasive lesions tended to have higher, and similar, beta-catenin concentrations (LG 0.99 RFU, HG dysplasia 1.04 RFU) than NL (0.68 RFU) or CA (0.76 RFU), a pattern similar to that seen in the immunohistochemistry. CONCLUSIONS: Beta-catenin expression is increased in precancerous squamous lesions of the esophagus and may be a useful early detection marker of esophageal disease. LOH is frequently found at polymorphic markers near this gene but mutations of this gene are rare. Other mechanisms such as those involved in protein stability and degradation need further investigation.

KEY WORDS: Esophagus, Squamous cell cancer, beta-catenin, proteomics, dysplasia.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Prognostic Markers.

This presentation received an honorable mention in our poster contest and was recognized with the Symposium Presidents' Award for Scientific Excellence.