Novel Breast Cancer Metastasis-Associated Proteins

Abstract

With the use of the breast cancer metastatic model, which comprises four isogenic cell lines, iTRAQ-based ESI-LC/MS/MS proteomics was employed to catalog protein expression changes as cancer cells acquire increasing metastatic potential. From more than 1000 proteins detected, 197 proteins, including drug-targetable kinases, phosphatases, proteases and transcription factors, displayed differential expression when cancer cells becomes more metastatic. Overall, the number of protein expression changes was evenly distributed across mildly ( approximately 30%), moderately ( approximately 40%) and aggressively ( approximately 30%) metastatic cancer cells. Some changes were found to be specific to one while others were required for two or more phenotypes. KEGG Orthology suggests major reprogramming in cell metabolism and to smaller extents in genetic and environmental information processing. Ten novel metastasis-associated proteins were identified and the iTRAQ-based expression profiles of 7 proteins were verified to be congruent with antibody-based methods. With the use of tissue microarrays comprising 50 matched cases of invasive and metastatic lesions, the expression profiles of SH3GLB1 and SUB1, SND1, TRIM28 were validated to be down- and up-regulated, respectively, during clinical progression of carcinoma in situ to invasive and metastatic carcinomas. Our study has unraveled proteome-wide molecular aberrations and potentially new players in breast cancer metastasis.

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells

“…A previous proteomic profile identified high SND1 expression in metastatic breast cancer cells and in tumor samples of metastatic breast cancer patients. 39 Moreover, microarray analyses indicated that depletion of SND1 in breast cancer cells leads to the down\regulation of genes associated with metastasis and chemoresistance. 40 SAM68 is also upregulated in breast cancer cells, and its expression correlates with malignant and aggressive phenotypes.…”

“…Primary antibodies used (1:1000 dilution; overnight at 4 1C) were the following: rabbit anti-ERK2, mouse antiMyc, rabbit anti-SAM68 and goat anti-lamin B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-tubulin, mouse anti-FLAG (Sigma-Aldrich) and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), mouse anti-RNAPII (CTD4H8; Millipore, Billerica, MA, USA) and mouse anti-SND1. 39 Immunoprecipitation experiments Nuclear extracts were used in co-immunoprecipitation experiments as previously described 23,58 using 2 mg of anti-SND1, anti-FLAG, anti-RNAPII or anti-SAM68 antibodies and either mouse or rabbit IgGs as control. After three washes with lysis buffer, proteins were eluted in sodium dodecyl sulphate sample buffer for western blot analysis.…”

Bif-1 suppresses breast cancer cell migration by promoting EGFR endocytic degradation

“…23 Moreover, a recent study using a mouse mammary tumor model revealed a decrease in Bif-1 expression as cells became more metastatic, suggesting a potential function for Bif-1 in breast cancer metastasis. 24 In this manuscript, we report a novel tumor suppressive function of Bif-1 in triple negative metastatic breast cancer. Knockdown of Bif-1 more acidic during their progression through the endocytic pathway in order to support the proper functioning of acid hydrolases.…”

Strategies and Challenges in Measuring Protein Abundance Using Stable Isotope Labeling and Tandem Mass Spectrometry

“…The disadvantages of this type of chemical labeling are the presence of potential side reactions, the extra steps required to remove excess reagents and derivatization byproducts resulting in difficulty in achieving complete labeling. The iTRAQ approach has been used for several large scale proteomic quantitative studies including time resolved monitoring of kinase reactions (Zhang et al 2005), comparison of organelle proteomes (Yan, Hwang, and Aebersold 2008) and monitoring of protein expression changes as cancer cells acquire increasing metastatic potential (Ho et al 2009). Combining quantitation with phosphoproteomics, Aebersold and colleagues (Pflieger et al 2008) recently described an iTRAQ method to simultaneously identify components and phosphorylation sites of protein complexes.…”