Differential Expression of Novel Tyrosine Kinase Substrates during Breast Cancer Development

Abstract

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ TM technology, and LC-MS/MS. Using complementary MALDI-and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.

Differential Protein Expression Profiling by iTRAQ-Two-dimensional LC-MS/MS of Human Bladder Cancer EJ138 Cells Transfected with the Metastasis Suppressor KiSS-1 Gene

“…Mass spectrometry techniques have become the dominant means of protein identification (20). The use of isobaric tags for relative and absolute quantitation (iTRAQ) combined with multidimensional liquid chromatography (LC) and tandem MS analysis (21) is emerging as a powerful methodology in the search for diseasespecific targets and biomarkers using cell lines (22)(23)(24)(25)(26)(27)(28)(29)(30), tissues (31)(32)(33)(34)(35)(36)(37), and body fluids (38,39). The iTRAQ method places tags on primary amines (NH 2 -terminal or -amino group of the lysine side chain), allowing detection of most tryptic peptides.…”

Phosphoproteomics for the Mapping of Altered Cell Signaling Networks in Breast Cancer

“…Many of these analyses have focused in tyrosine phosphorylation profiles due to the fact that approximately half of the tyrosine kinase complement of the human kinome is implicated in human cancers [4], and provides important targets for cancer treatment, as well as biomarkers for patient stratification. Recently, Chen et al adapted LC-MS/MS technology to assess the tyrosine phosphorylation profile in the MCF10AT model of breast cancer progression [25]. This study identified and validated seven proteins, termed SPAG9, CYFIP1, RPS2, TOLLIP, SLC4A7, WBP2, and NSFLC1, to be authentic tyrosine kinase substrates.…”

“…In contrast, only 25% of the samples showed increased levels of TOLLIP when normal cells become cancerous. Moreover detection of aberrant expression of TOLLIP and SLC4A7 in pre-neoplastic lesions suggests that they represent potential biomarkers that could complement mammography and histopathology for screening and early detection of breast cancer [25].…”

Polygenic Susceptibility to Breast Cancer: Current State-of-The-Art

“…The function of NEK10 is unknown, but evidence shows that NEK2, 6, 7 and 9 are involved in mitosis [75]. SLC4A7 is a potential tyrosine kinase substrate where expression is reduced in breast cancer tumors, and is predicted to affect the pH of the microenvironment around breast tumor cells [76].…”