Selective Tyrosine Hyperphosphorylation of Cytoskeletal and Stress Proteins in Primary Human Breast Cancers

Abstract

Purpose: Small-molecule growth factor receptor inhibitors block cell growth in vitro and downstream signaling in vivo, but controlled trials in patients with advanced solid tumors have yielded disappointing response rates. To clarify this discrepancy, we compared the patterns of tyrosine phosphoprotein expression in human cancer cells and primary tumors.Experimental Design: Immunoaffinity chromatography, two-dimensional electrophoresis, and antiphosphotyrosine immunoblotting were combined with mass spectrometry to determine the phosphoproteomic signatures of 40 matched normal and malignant tissues from patients with breast or liver cancer. The identities and abundance of the detected tyrosine phosphoproteins were compared with those of ligand-responsive A431 cells.Results: Patterns of tyrosine-phosphorylated proteins are similar among normal tissues of the same origin but vary markedly between different tissues. Primary breast tumors exhibit a strikingly homogeneous tyrosine phosphorylation profile, whereas liver cancers display greater phosphoproteomic diversity. The main breast-tumor-specific tyrosine phosphoproteins are cytoskeletal molecules (actin, tubulin, and vimentin) and molecular chaperones (Hsp70, Hsc71, and Grp75). In contrast, control studies in ligand-stimulated A431 human cancer cells revealed an additional phosphorylated subset of promitogenic phosphoproteins (Grb2, Shc, Jnk2, phospholipase C-␥, and phosphatidylinositol 3-kinase).Conclusions: Identification of cytoskeletal and stress proteins as the most abundant tyrosine phosphoproteins in breast tumors implicates these molecules, rather than promitogenic effectors, as the prime stoichiometric substrates for kinase-inhibitory anticancer drugs in vivo. Because phosphorylated cytoskeletal proteins and chaperones mediate cell motility and apoptotic resistance, respectively, these data raise the intriguing possibility that small-molecule tyrosine kinase inhibitors may be of greatest value either as adjuvant antimetastatic/-invasive drugs or as chemo-/radiosensitizers.

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

“…This is in agreement with the finding that different GPCR agonists, including bradykinin and serotonin, induce remodeling of the vimentin intermediate filament network through phosphorylation events [27,32,37]. While tyrosine phosphorylation of vimentin has been detected in cancer cells and putatively linked to migration and invasion [38], little is known about the specific function of vimentin phosphotyrosines. Conversely, vimentin phosphorylation of serine residues has been better characterized and shown to induce the interaction with docking proteins, to trigger fiber disassembly and ultimately to promote cell migration [27].…”

“…These phosphorylation events are triggered by a wide range of protein kinases, such as PKA and PKC [15,19], PKG [20], Rho-kinase and Aurora B [21], PAK [22,23], MAPKAP K-2 [10], Cdc2 kinase [24] and CaMKII [25] and involve a large number of serine residues (e.g. Ser 4,[6][7][8][9]25,33,38,41,50,55,65,71,72,82) with a complex pattern of overlapping specificities [15,26,27].…”

“…These phosphorylation events are triggered by a wide range of protein kinases, such as PKA and PKC [15,19] [25] and involve a large number of serine residues (e.g. Ser 4,[6][7][8][9]25,33,38,41, 50, 55, 65, 71, 72, 82) with a complex pattern of overlapping specificities [15,26,27].Although vimentin appears involved in multiple cellular processes, mice lacking this protein are viable and do not show overt phenotypes [28]. Nonetheless, fibroblasts from vimentindeficient mice show mechanical instability and impaired ability to migrate and to contract a 3-D collagen network [29], thus providing a genetic evidence for the involvement of vimentin in cell plasticity and motility [30,31].…”

Heat Shock Protein 70 Is Secreted from Tumor Cells by a Nonclassical Pathway Involving Lysosomal Endosomes

“…8A). HSP70 can be phosphorylated under some circumstances and may be ubiquitinated on association with the ubiquitin ligase CHIP (42,43). Translocation of a fraction of HMGB1 and engrailed-2 to the secretory pathway involves hyperacetylation and dephosphorylation, respectively (44,45).…”

Mining the Tumor Phosphoproteome for Cancer Markers

“…The hypothesis is supported by the observation that ErbB2/Her-2 gene amplification can be acquired as breast cancer progresses (42). The evidence for the existence of tumor-specific phosphoproteome came from our novel finding that the phosphotyrosine proteome of the breast and liver tumors were indeed distinct (43). This provided a proof of principle that it is possible to mine the tumor phosphoproteome for potential biomarkers.…”