Discovery of Novel Kidney Cancer Biomarkers by Quantitative Proteomic Approaches

Most of the cancers are diagnosed at advanced stages, which lead to high mortality rate of cancer. In order to lead a healthy life, the best way to fight cancer is to detect it earlier. Diagnosis of cancer by checking tumor markers in body fluids is the most convenient way clinically for early diagnosis of cancer. Around 208,500 new cases of kidney cancer are diagnosed in the world each year, accounting for 2~3% of all cancers. In the United States in 2008, kidney cancer is estimated to cause 54,390 new cases and 13,010 deaths. There are several types of kidney cancer. Two most common ones are renal cell carcinoma and transitional cell carcinomas. Kidney cancer does not always cause symptoms, but if there are symptoms they can include haematuria, high blood pressure, persistent low back pain, and a mass or lump detected in the abdominal area. However, many of these symptoms are non-specific and may relate to a non-cancerous kidney or urinary tract problem. There is a compelling need for reliable tumor markers for renal cancer. One promising approach to meet this clinical need is to study the urine and tissue proteome during the occurrence of disease. There is a growing consensus that effective and accurate detection of early stage cancer will likely rely on marker panels that have greater specificity and sensitivity than each marker alone. Recently, multiple reaction monitoring (MRM) coupled with stable isotope dilution mass spectrometry (MS) for direct quantification of proteins in cell lysates as well as body fluids has shown considerable promise. Up to tens of candidate proteins can be nearly simultaneously targeted and quantified in clinical specimens. This technology is suitable for use in preclinical studies to rapidly screen large numbers of candidate protein biomarkers in the hundreds of patient samples necessary for verification. In the past two years, we have established a urine sample preparation workflow for the iTRAQ (isobaric tag for relative and absolute quantitation) technique in differential proteomics study. We have applied this technology platform to discover proteins that were differentially expressed between pooled urine samples from non-tumor controls and three bladder cancer patient subgroups with different grades/stages. Combining the results of two independent clinical sample sets, a total of 638 urine proteins were identified. Among them, 55 dysregulated proteins consistently showed more than two-fold differences in both sample sets. These proteins represent potential bladder cancer biomarkers. Western blot analyses of individual urine samples confirmed that the levels of four proteins were significantly elevated in urine samples from bladder cancer patients. Finally, we quantified a novel protein (BC-1) in urine samples using a commercial ELISA and confirmed again its potential diagnostic value (94.6% sensitivity and 92.0% specificity at a cutoff value of 11.16 ng/mL) and early detection (83.8% sensitivity and 94.0% specificity at a cutoff value of 16.30 ng/mL) (Chen et al., submitted to Mol. Cell. Proteomics). Although above biomarkers were discovered from urine specimens of bladder cancer, they may present the dysfunction of one or more organs in the urinary tract. This means that we should verify those discovered bladder cancer biomarkers in urine from more types of urological diseases, such as urinary tract infection and renal cancer, to clarify their relevance with urinary tract organs. On the basis of the results described above, we attempt to address the following issues in this 4-year proposal: (1) Validation of discovered urinary biomarkers of bladder cancer in different types of urological diseases including kidney cancer. This part of work will elucidate the clinical significance and relevance of these molecules in renal cancer. (2) Using bladder cancer patients and age-matched volunteers as control groups to discover novel and disease-specific urine/tissue proteins associated with renal cancer by comparative proteomics approaches for non-invasive detection and mechanistic elucidation of tumorgenesis. (3) Development of antibody-based and MRM-MS-based (without using any antibody), multiplexed assays to absolutely quantify renal cancer biomarkers that discovered in this project for the purpose of high-throughput screening of renal cancer. The first issue of the project will be investigated in the first year of this four-year project. After the collection of urine and tumor specimens in the first year, we will start to perform the quantitative proteomic studies using pooled urine and laser capture micro-dissected tissue specimens of renal cancer patients in the second and third years to address the second issue. The differentially-expressed urine or tissue proteins associated with kidney cancer will be further validated by different assays in urine and tissue specimens of a large number of individual samples during the third and fourth project years. The information and technology platforms generated and developed from this proposal will be valuable for early detection and diagnosis of kidney cancer in clinic.

Project ID:PG10201-0145
External Project ID:NHRI-EX102-10015BI