ErbB2 Receptor in Breast Cancer: Implications in Cancer Cell Migration, Invasion and Resistance to Targeted Therapy

Overexpression of ErbB2 is found in several types of human carcinomas. In breast tumors, ErbB2 overexpression is detected in up to 20% of patients. Breast cancers in with amplification of ErbB2 are characterized by rapid tumor growth, lower survival rate and increased disease progression. The molecular mechanisms underlying the oncogenic action of ErbB2 involve a complex signaling network that tightly regulates malignant cell migration and invasion and hence metastatic potential. Recent efforts have been made to identify gene expression signatures of ErbB2-positive invasive breast cancers that may represent important mediators of ErbB2-induced tumorigenesis and metastatic progression. In this chapter, we will discuss the canonical ErbB2 signaling pathways responsible for tumor growth and dissemination along with newly identified mediators such as adaptor protein p130Cas and miRNAs. From a therapeutic point of view, the treatment with anti-ErbB2 monoclonal antibody trastuzumab has greatly improved the outcomes of patients with ErbB2 aggressive cancer. Nevertheless, de novo and acquired resistance to trastuzumab therapy still represent a major clinical problem. In the second part of the chapter, we will provide an overview of the mechanisms so far implicated in the onset of resistance to targeted therapy and of the new strategies to overcome resistance

Cas proteins: dodgy scaffolding in breast cancer

The members of the Cas protein family (p130Cas/ BCAR1, Nedd9/HEF1, EFS and CASS4) are scaffold proteins required for the assembly of signal transduction complexes in response to several stimuli, such as growth factors, hormones and extracellular matrix components. Given their ability to integrate and coordinate multiple signalling events, Cas proteins have emerged as crucial players in the control of mammary cell proliferation, survival and differentiation. More importantly, it has been found that alterations of their expression levels result in aberrant signalling cascades, which promote initiation and progression of breast cancer. Based on the increasing data from in vitro, mouse model and clinical studies, in this review we will focus on two Cas proteins, p130Cas/BCAR1 and Nedd9, and their coupled signalling pathways, to examine their role in mammary cell transformation and in the acquirement of invasiveness and drug resistance of breast cancer cells

p130Cas/ BCAR1 and p140Cap/ SRCIN1 Adaptors: The Yin Yang in Breast Cancer?

p130Cas/BCAR1 is an adaptor protein devoid of any enzymatic or transcriptional activity, whose modular structure with various binding motifs, allows the formation of multi-protein signaling complexes. This results in the induction and/or maintenance of signaling pathways with pleiotropic effects on cell motility, cell adhesion, cytoskeleton remodeling, invasion, survival, and proliferation. Deregulation of p130Cas/BCAR1 adaptor protein has been extensively demonstrated in a variety of human cancers in which overexpression of p130Cas/BCAR1 correlates with increased malignancy. p140Cap (p130Cas associated protein), encoded by the SRCIN1 gene, has been discovered by affinity chromatography and mass spectrometry analysis of putative interactors of p130Cas. It came out that p140Cap associates with p130Cas not directly but through its interaction with the Src Kinase. p140Cap is highly expressed in neurons and to a lesser extent in epithelial tissues such as the mammary gland. Strikingly, in vivo and in vitro analysis identified its tumor suppressive role in breast cancer and in neuroblastoma, showing an inverse correlation between p140Cap expression in tumors and tumor progression. In this review, a synopsis of 15 years of research on the role of p130Cas/BCAR1 and p140Cap/SRCIN1 in breast cancer will be presented

Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry Reveals Stimulation-dependent Multisite Phosphorylation

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes

Sguardi su Aurora: tra centro e periferia

Sintesi delle indagini svolte da AuroraLAB nel primo anno di attività. Fornisce alcune fotografie del territorio, delle sue caratteristiche, delle sue risorse, delle criticità

p130Cas promotes invasiveness of three dimensional ErbB2-transformed mammary acinar structures by enhanced activation of mTOR/p70S6K and Rac1

ErbB2 over-expression is detected in approximately 25% of invasive breast cancers and is strongly associated with poor patient survival. We have previously demonstrated that p130Cas adaptor is a crucial mediator of ErbB2 transformation. Here, we analysed the molecular mechanisms through which p130Cas controls ErbB2-dependent invasion in three-dimensional cultures of mammary epithelial cells. Concomitant p130Cas over-expression and ErbB2 activation enhance PI3K/Akt and Erk1/2 MAPK signalling pathways and promote invasion of mammary acini. By using pharmacological inhibitors, we demonstrate that both signalling cascades are required for the invasive behaviour of p130Cas over-expressing and ErbB2 activated acini. Erk1/2 MAPK and PI3K/Akt signalling triggers invasion through distinct downstream effectors involving mTOR/p70S6K and Rac1 activation, respectively. Moreover, in silico analyses indicate that p130Cas expression in ErbB2 positive human breast cancers significantly correlates with higher risk to develop distant metastasis, thus underlying the value of the p130Cas/ErbB2 synergism in regulating breast cancer invasion. In conclusion, high levels of p130Cas favour progression of ErbB2-transformed cells towards an invasive phenotype