An Approach to Studying Lung Cancer-related Proteins in Human Blood

Shu Guo ,Husheng Zhang,Ning Han ,Wuju Li,Jie He,Di Xia ,Xiaohong Qian,Yan Liu,Mengge Gao ,Jinfang Ma,Yanning Gao,Lei Li,Yun Cai,Liyan Jiao,Min Li,Ting Xiao,Dongmei Lin,Kai Su,Zhi Hu,Dechao Zhang,Ying Ma,Na Xu ,Susheng Shi,Shujun Cheng,Kaitai Zhang,Jinyun Yuan ,Wantao Ying,Hongwei Zheng

doi:10.1074/mcp.m500055-mcp200

Abstract

Early stage lung cancer detection is the first step toward successful clinical therapy and increased patient survival. Clinicians monitor cancer progression by profiling tumor cell proteins in the blood plasma of afflicted patients. Blood plasma, however, is a difficult cancer protein assessment medium because it is rich in albumins and heterogeneous protein species. We report herein a method to detect the proteins released into the circulatory system by tumor cells. Initially we analyzed the protein components in the conditioned medium (CM) of lung cancer primary cell or organ cultures and in the adjacent normal bronchus using one-dimensional PAGE and nano-ESI-MS/MS. We identified 299 proteins involved in key cellular process such as cell growth, organogenesis, and signal transduction. We selected 13 interesting proteins from this list and analyzed them in 628 blood plasma samples using ELISA. We detected 11 of these 13 proteins in the plasma of lung cancer patients and non-patient controls. Our results showed that plasma matrix metalloproteinase 1 levels were elevated significantly in late stage lung cancer patients and that the plasma levels of 14-3-3 sigma, beta, and eta in the lung cancer patients were significantly lower than those in the control subjects. To our knowledge, this is the first time that fascin, ezrin, CD98, annexin A4, 14-3-3 sigma, 14-3-3 beta, and 14-3-3 eta proteins have been detected in human plasma by ELISA. The preliminary results showed that a combination of CD98, fascin, polymeric immunoglobulin receptor/secretory component and 14-3-3 eta had a higher sensitivity and specificity than any single marker. In conclusion, we report a method to detect proteins released into blood by lung cancer. This pilot approach may lead to the identification of novel protein markers in blood and provide a new method of identifying tumor biomarker profiles for guiding both early detection and therapy of human cancer.

Highlights

Stage lung cancer detection is the first step toward successful clinical therapy and increased patient survival
Proteome Database of conditioned medium (CM) Proteins—We used proteomic techniques to analyze the CM samples harvested from the primary cultures of lung cancer cells and the adjacent normal bronchial epithelial cells of six lung cancer patients
After separating the proteins with 1-D SDS-PAGE (Fig. 1), digesting them in-gel, extracting the peptides, and analyzing them with nano-LC-ESI-MS/MS, we identified 231 distinct proteins (Supplemental Table 5). These proteins are involved in cell growth and/or maintenance, protein metabolism, catabolism, response to external stimulus, organogenesis, signal transduction, and cell adhesion activities

Summary

Introduction

Stage lung cancer detection is the first step toward successful clinical therapy and increased patient survival. In this study we analyzed the proteins released into serum-free conditioned medium (CM)1 from the tumor microenvironment with short time-cultured lung cancer and adjacent normal bronchial epithelial cells, one-dimensional (1-D) PAGE, and liquid chromatography-tandem mass spectrometry.

Results

Conclusion