Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Abstract

IntroductionBreast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.MethodWe performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).ResultsUnbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.ConclusionsOur results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.

Early biomarker discovery in urine of Walker 256 subcutaneous rat model

“…It was interesting that the pathways that changed in this experiment were similar to the pathways that were enriched in a previous urinary proteomics study in breast cancer patients [6]. The pathways included acute phase response signaling, LXR/RXR activation, production of nitric oxide and ROS in macrophages, IL-12 signaling and production in macrophages, and clathrin-medicated endocytosis signaling.…”

“…In recent years, advances in proteomics, especially in mass spectrometry, have led to the identification of more than 3000 unique proteins in human urine. Meanwhile, urinary proteomics has been successfully applied to discover novel biomarkers for cancer diagnosis and cancer monitoring [4][5][6]. However, whether urine protein biomarkers assist in the early diagnosis of cancer is unclear.…”

Early urinary candidate biomarker discovery in a rat thioacetamide-induced liver fibrosis model

“…However, urine accumulates changes 55 of the body, which makes it a better early biomarker source [22,23] . Urinary proteomics has 56 become increasingly important in studies of quantitative changes in proteins resulting 57 from changes in disease states [24][25][26][27][28] ; moreover, numerous urinary protein biomarkers 58 have been reported in different diseases [29][30][31][32] . These findings suggest that urinary proteins 59 may serve as non-invasive biomarkers for diseases.…”

Quantitative body fluid proteomics in medicine — A focus on minimal invasiveness

“…Under normal conditions, urine contains proteins originating from the blood and kidneys [134][135][136][137][138][139], making urine a good source for analysis of diseases affecting the kidney or the urogenital tract; such as kidney failure resulting from high blood pressure and diabetic nephropathy [140,141], prostate cancer [142,143], polycystic kidney disease [144], kidney chronic allograft dysfunction [145], chronic allograft nephropathy [146], congenital obstructive nephropathy [147], lupus nephritis [148], urolithiasis [149], in addition to urinary, renal and bladder cancer [150][151][152][153][154][155][156][157][158][159][160][161]. Besides urogenital and kidney dysfunctions, urinary proteomics has a great potential in biomarker studies of coronary artery atherosclerosis [162,163], obstructive sleep apnea [164], ovarian cancer [165], breast cancer [166] and sepsis [167,168].…”

“…Urine proteome analysis may potentially unravel markers for cancers of urogenital or systemic origin including bladder [150][151][152][153][154]156,157,160,161], prostate [170], renal [158,159], breast [166] and ovarian cancers [165] (Table 7). There has been an increasing interest in developing urine biomarkers for the detection of renal allograft rejection as an alternative to percutaneous needle biopsy, which is costly and associated with significant patient morbidity and mortality [79].…”