Gillian M. Lehrbach

Tyrosine phosphorylation profiling reveals the signaling network characteristics of basal breast cancer cells

To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry-based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e.g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup.

Aberrant expression of cortactin in head and neck squamous cell carcinoma cells is associated with enhanced cell proliferation and resistance to the epidermal growth factor receptor inhibitor gefitinib

The CTTN gene (formerly designated EMS1), encodes cortactin, a key regulator of dynamic actin networks. Both CTTN and CCND1, the latter encoding the cell cycle regulator cyclin D1, reside at chromosomal locus 11q13, a region commonly amplified in breast cancers and head and neck squamous cell carcinoma (HNSCC). Previously, we identified a novel role for cortactin in cancer cells, whereby cortactin overexpression attenuated ligand-induced down-regulation of the epidermal growth factor (EGF) receptor (EGFR), leading to sustained signaling. However, how this affected growth factor-induced cellular responses was unclear. Here, by modulation of cortactin expression in a panel of HNSCC cell lines, we show that cortactin overexpression enhances serum- and EGF-stimulated proliferation under both anchorage-dependent and anchorage-independent conditions and also increases resistance to anoikis (detachment-induced apoptosis). These effects are associated with increased activation of extracellular signal-regulated kinase and/or AKT. Furthermore, we report that cortactin stabilizes the c-MET receptor tyrosine kinase and enhances hepatocyte growth factor-induced mitogenesis and cell scattering. Therefore, cortactin may modulate signaling by a broader range of receptors than originally proposed and thereby affect a variety of responses. Finally, we have determined that cortactin overexpression, either alone or in combination with cyclin D1 up-regulation, promotes resistance to the EGFR kinase inhibitor gefitinib. These findings indicate that cortactin may play multiple roles in progression of HNSCC and should be evaluated as a marker of prognosis, disease progression, and therapeutic responsiveness, particularly to EGFR-directed agents.

Phosphorylation‐dependent binding of 14‐3‐3 terminates signalling by the Gab2 docking protein

Grb2‐associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor‐induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14‐3‐3 proteins. Together, these events mediate negative‐feedback regulation, as Gab2S210A/T391A exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14‐3‐3‐binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site‐selective binding of 14‐3‐3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14‐3‐3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines.

The plasma membrane Ca(2+) ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca(2+) plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca(2+) regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.

The E3 Ubiquitin Ligase EDD Regulates S-Phase and G2/M DNA Damage Checkpoints

The cellular response to DNA damage is critical for maintenance of genomic integrity and inhibition of tumorigenesis. Mutations or aberrant expression of the E3 ubiquitin ligase EDD have been observed in a number of carcinomas and we recently reported that EDD modulates activity of the DNA damage checkpoint kinase, CHK2. Here, we demonstrate that EDD is necessary for G1/S and intra S phase DNA damage checkpoint activation and for the maintenance of G2/M arrest after double strand DNA breaks. Defective checkpoint activation in EDD-depleted cells led to radio-resistant DNA synthesis, premature entry into mitosis, accumulation of polyploid cells, and cell death via mitotic catastrophe. In addition to decreased CHK2 activation in EDD-depleted cells, the expression of several key cell cycle mediators including Cdc25A/C and E2F1 was altered, suggesting that these checkpoint defects may be both CHK2-dependent and -independent. These data support a role for EDD in the maintenance of genomic stability, emphasising the potential importance of dysregulated EDD expression and/or function in the evolution of cancer.

A novel plant toxin, persin, with in vivo activity in the mammary gland, induces Bim-dependent apoptosis in human breast cancer cells

Phytochemicals have provided an abundant and effective source of therapeutics for the treatment of cancer. Here we describe the characterization of a novel plant toxin, persin, with in vivo activity in the mammary gland and a p53-, estrogen receptor–, and Bcl-2-independent mode of action. Persin was previously identified from avocado leaves as the toxic principle responsible for mammary gland–specific necrosis and apoptosis in lactating livestock. Here we used a lactating mouse model to confirm that persin has a similar cytotoxicity for the lactating mammary epithelium. Further in vitro studies in a panel of human breast cancer cell lines show that persin selectively induces a G2-M cell cycle arrest and caspase-dependent apoptosis in sensitive cells. The latter is dependent on expression of the BH3-only protein Bim. Bim is a sensor of cytoskeletal integrity, and there is evidence that persin acts as a microtubule-stabilizing agent. Due to the unique structure of the compound, persin could represent a novel class of microtubule-targeting agent with potential specificity for breast cancers. [Mol Cancer Ther 2006;5(9):2300–9]

Progestins Reinitiate Cell Cycle Progression in Antiestrogen-Arrested Breast Cancer Cells through the B-Isoform of Progesterone Receptor

Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)-negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH(2)-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest.

Identification of PUMA as an estrogen target gene that mediates the apoptotic response to tamoxifen in human breast cancer cells and predicts patient outcome and tamoxifen responsiveness in breast cancer

Recognition of the pivotal role of estrogen in the aetiology of breast cancer has led to the development of antiestrogens (AE), such as tamoxifen (TAM) as effective therapies for the treatment and prevention of this disease. However, despite their widespread clinical efficacy, response to AEs is often short-lived, and acquired or innate therapeutic resistance remains a major obstacle in the successful treatment of breast cancer. Thus, delineating the intracellular pathways that mediate the cellular response to estrogen could potentially lead to new, more effective approaches to the treatment of breast cancer, particularly endocrine-resistant disease. Here, we have identified the BCL-2 homology 3 (BH3)-only, pro-apoptotic regulator, PUMA (p53 upregulated modulator of apoptosis) as an estrogen target gene that is acutely downregulated in response to estrogen in breast cancer cell lines, independently of their p53 status. PUMA is transcriptionally upregulated following treatment with TAM, and knock down of PUMA expression in these cells attenuates the apoptotic response to TAM. Furthermore, low PUMA expression in breast carcinomas is significantly associated with breast cancer-specific death (P=0.0014 and P=0.0115, for mRNA and protein, respectively), and worse outcome in TAM-treated patients (mRNA, P=1.49e-05). These findings suggest that the dysregulation of apoptotic signaling pathways such as those executed through PUMA, can significantly impact on both the progression and therapeutic responsiveness of breast cancer. Moreover, they provide a convincing rationale for exploring new therapeutic approaches involving endocrine and non-endocrine therapies that target apoptotic pathways as an effective strategy for tackling endocrine refractory disease.