Sarah J. Parsons

STAT5b, a Mediator of Synergism between c-Src and the Epidermal Growth Factor Receptor

Overexpression of the epidermal growth factor receptor (EGFR) and its association with the tyrosine kinase, c-Src, is correlated with increased cellular proliferation and tumorigenesis. Previous studies have shown that EGFR and c-Src co-overexpression and association leads to the c-Src-mediated phosphorylation of tyrosine 845 of the EGFR and that mutation of Tyr845 ablates epidermal growth factor (EGF)-induced DNA synthesis. Here, we investigate the contribution of the signal transducers and activators of transcription (STAT5b) in the signaling pathways regulated by EGFR and c-Src overexpression in human breast tumor cell lines as well as in a mouse fibroblast model (C3H10T1/2). We demonstrate that 1) activation of STAT5b by EGF requires overexpression of the EGFR, 2) co-overexpression of c-Src alone does not result in EGF-induced activation of STAT5b but enhances that seen in EGFR-overexpressing cells, and 3) EGF-induced tyrosine phosphorylation of STAT5b requires Tyr845 of the EGFR. Furthermore, the stable overexpression of a kinase-defective c-Src in the context of EGFR overexpression results in a decrease in the tyrosine phosphorylation of STAT5b in response to EGF and a more dramatic decrease in EGF-induced transcriptional activation of STAT5b, suggesting an integral role for c-Src in the physiological actions of STAT5b. Using a dominant negative STAT5b, we provide evidence that one such physiological action is to mediate EGF-induced DNA-synthesis. Finally, the use of site-specific tyrosine mutants demonstrates that EGF-induced phosphorylation of STAT5b involves not only tyrosine 699 of STAT5b, which is required for its transcriptional activation, but also three previously identified tyrosines in the C terminus of STAT5b (Tyr725/Tyr740/Tyr743).

p190RhoGAP is cell cycle regulated and affects cytokinesis

p190RhoGAP (p190), a Rho family GTPase-activating protein, regulates actin stress fiber dynamics via hydrolysis of Rho-GTP. Recent data suggest that p190 also regulates cell proliferation. To gain insights into the cellular process(es) affected by p190, we altered its levels by conditional or transient overexpression. Overexpression of p190 resulted in a multinucleated phenotype that was dependent on the GTPase-activating protein domain. Confocal immunofluorescence microscopy revealed that both endogenous and exogenous p190 localized to the newly forming and contracting cleavage furrow of dividing cells. However, overexpression of p190 resulted in abnormal positioning of the furrow specification site and unequal daughter cell partitioning, as well as faulty furrow contraction and multinucleation. Furthermore, levels of endogenous p190 protein were transiently decreased in late mitosis via an ubiquitin-mediated degradation process that required the NH2-terminal GTP-binding region of p190. These results suggest that a cell cycle–regulated reduction in endogenous p190 levels is linked to completion of cytokinesis and generation of viable cell progeny.

Epidermal growth factor-receptor activation modulates Src-dependent resistance to lapatinib in breast cancer models

IntroductionSrc tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib.MethodsTo determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses.ResultsSrc activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling.ConclusionsComplete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.

p190RhoGAP negatively regulates Rho activity at the cleavage furrow of mitotic cells

Previous studies demonstrated that p190RhoGAP (p190) negatively affects cytokinesis in a RhoGAP-dependent manner, suggesting that regulation of Rho may be a critical mechanism of p190 action during cytokinesis. P190 localizes to the cleavage furrow (CF) of dividing cells, and its levels decrease during late mitosis by an ubiquitin-mediated mechanism, consistent with the hypothesis that high RhoGTP levels are required for completion of cytokinesis. To determine whether RhoGTP levels in the CF are affected by p190 and to define the phase(s) of cytokinesis in which p190 is involved, we used FRET analysis alone or in combination with time-lapse microscopy. In normal cell division activated Rho accumulated at the cell equator in early anaphase and in the contractile ring, where it co-localized with p190. Real-time movies revealed that cells expressing elevated levels of p190 exhibited multiple cycles of abnormal CF site selection and ingression/regression, which resulted in failed or prolonged cytokinesis. This was accompanied by mislocalization of active Rho at the aberrant CF sites. Quantified data revealed that in contrast to ECT2 and dominate negative p190 (Y1283Ap190), which resulted in hyper-activated Rho, Rho activity in the CF was reduced by wild type p190 in a dose-dependent manner. These results suggest that p190 regulates cytokinesis through modulation of RhoGTP levels, thereby affecting CF specification site selection and subsequent ring contraction.

MMTV-EGF receptor transgene promotes preneoplastic conversion of multiple steroid hormone-responsive tissues.

Correlative analyses of tumors and patient‐derived cell lines of the human reproductive system suggest that overexpression of EGF contributes to the oncogenic phenotype. However, it is unclear at what stage in disease overexpression of the EGFR is most critical. To assess its role as an initiator of reproductive tissue tumor development, transgenic mice were derived with mouse mammary tumor virus (MMTV)‐regulated overexpression of the human EGFR. Although elevated expression of the EGFR in hormonally responsive tissues was observed, only one EGFR transgenic mouse developed a visible tumor over a 2‐year period. However, of 12 females monitored over the same time, hyperplasia, hypertrophy, or slight dysplasia was found in mammary glands of 55% of the animals examined, in the uterus or uterine horn of 89%, and in ovaries or oviducts of 100%. None of the reproductive tissues of the male transgenic animals or age‐matched, normal mice displayed these changes. These results revealed a role for the EGFR in the initiation of ovarian and uterine cancer and supported previous studies in breast cancer that the receptor can contribute to the neoplastic process in a significant albeit incremental way. J. Cell. Biochem. 103: 2010–2018, 2008.

Src inhibitors act through different mechanisms in Non-Small Cell Lung Cancer models depending on EGFR and RAS mutational status

Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non-Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs.

The Tumor Suppressor, p190RhoGAP, Differentially Initiates Apoptosis and Confers Docetaxel Sensitivity to Breast Cancer Cells

p190RhoGAP (p190) is a negative regulator of RhoGTPases and a putative tumor suppressor, whose mechanism of tumor suppression is poorly defined. Ectopic expression of p190 induces various morphological phenotypes, including multinucleation, dendrite-like formation, and chromatin condensation, suggesting an involvement in apoptosis. We examined the possibility that p190 can function as a tumor suppressor by regulating induction of apoptosis. We show that the predominant phenotype of p190 overexpression in a variety of cell lines is apoptosis, which is mediated through p190s regulation of Rho and caspases. The secondary phenotypes, multinucleation and dendrite-like formation, are determined by transformation status, not cell lineage, and appear to be intermediate phenotypes in the p190-induced apoptotic pathway. Finally, we show that p190 levels can regulate the apoptotic response of breast cancer cell lines to docetaxel through its regulation of Rho. Together, these findings suggest that one mechanism by which p190 can mediate its tumor-suppressive function is through regulation of Rho-activated cell death pathways and that this function can be exploited to optimize the action of cytoskeletal-based chemotherapeutics, such as the taxanes.

The Human Papillomavirus E7 Proteins Associate with p190RhoGAP and Alter Its Function

ABSTRACT Using mass spectrometry, we identified p190RhoGAP (p190) as a binding partner of human papillomavirus 16 (HPV16) E7. p190 belongs to the GTPase activating protein (GAP) family and is one of the primary GAPs for RhoA. GAPs stimulate the intrinsic GTPase activity of the Rho proteins, leading to Rho inactivation and influencing numerous biological processes. RhoA is one of the best-characterized Rho proteins and is specifically involved in formation of focal adhesions and stress fibers, thereby regulating cell migration and cell spreading. Since this is the first report that E7 associates with p190, we carried out detailed interaction studies. We show that E7 proteins from other HPV types also bind p190. Furthermore, we found that conserved region 3 (CR3) of E7 and the middle domain of p190 are important for this interaction. More specifically, we identified two residues in CR3 of E7 that are necessary for p190 binding and used mutants of E7 with mutations of these residues to determine the biological consequences of the E7-p190 interaction. Our data suggest that the interaction of E7 with p190 dysregulates this GAP and alters the actin cytoskeleton. We also found that this interaction negatively regulates cell spreading on a fibronectin substrate and therefore likely contributes to important aspects of the HPV life cycle or HPV-induced tumorigenesis. IMPORTANCE This study identifies p190RhoGAP as a novel cellular binding partner for the human papillomavirus (HPV) E7 protein. Our study shows that a large number of different HPV E7 proteins bind p190RhoGAP, and it identifies regions in both E7 and p190RhoGAP which are important for the interaction to occur. This study also highlights the likelihood that the E7-p190RhoGAP interaction may have important biological consequences related to actin organization in the infected cell. These changes could be an important contributor to the viral life cycle and during progression to cancer in HPV-infected cells. Importantly, this work also emphasizes the need for further study in a field which has largely been unexplored as it relates to the HPV life cycle and HPV-induced transformation.

INTERACTIONS OF STATs WITH SRC FAMILY KINASES

C-Src tyrosine kinase is the cellular homologue of v-Src, the oncogenic form of the protein encoded by the chicken retrovirus, Rous sarcoma virus (reviewed in 1, 2). The Src family of tyrosine kinases includes Src, Yes, Yrk, and Fyn which are ubiquitously expressed, and Hck, Fgr, Lyn, Lck and Blk which are expressed predominantly in hematopoetic cells (myeloid cells and B and T lymphocytes). These proteins are approximately 60kDa in molecular weight and are composed of six domains (Figure 1). For Src, these domains are: 1) the N-terminal domain that contains a signal for myristolyation and targets the cytosolic kinase to intracellular membranes; 2) a unique domain that is likely involved in protein-protein interactions; 3) an SH3 domain that binds to proline-rich sequences; 4) an SH2 domain that binds phosphotyrosine residues; 5) the catalytic kinase domain; and 6) the negative regulatory region at the C-terminus which includes Tyr530 (originally identified in chicken c-Src as Tyr527). Tyr530 is phosphorylated by C-terminal Src kinase (Csk), resulting in an intramolecular interaction between this phosphotyrosine and the SH2 domain of Src, thus rendering the kinase catalytically inactive. Displacement of phosphorylated Tyr530 by binding of another tyrosine phosphorylated protein, dephosphorylation, or mutation/deletion of Tyr530 (as in v-Src) changes the conformation of c-Src, resulting in catalytic activation. The Src family of kinases plays an integral role in growth factor-induced proliferation as well as the initiation and progression of many human cancers, and differentiation (1, 3, 4). This chapter will review the current knowledge regarding interactions between c-Src (and family members) and the STAT proteins, including the association with and phosphorylation of STATs by Src family kinases (SFKs), the role of SFKs in the activation of STATs by growth factors, and finally, biological effects of SFKs that require STAT activation.

Convergence of EGF Receptor and Src Family Signaling Networks in Cancer

EGF receptor (EGFR) and c-Src are tyrosine kinases of the receptor and non-receptor classes, respectively, that which are frequently co-overexpressed or co-activated in multiple human cancers, including those of breast, prostate, lung, and colon. Most of these cancers express non-mutated forms of each kinase, and overexpression of either is weakly or non-oncogenic. However, When co-overexpressed, however, they exhibit profound synergism that up-regulates many neoplastic processes, including cell proliferation, survival, and metastasis. This synergism is dependent upon or greatly enhanced by physical association between c-Src and ligand-stimulated EGFR, which leads to activation of both kinases, phosphorylation of EGFR by c-Src, and enhanced phosphorylation of EGFR and c-Src substrates. Non-EGFR ligands, such as agonists for G-protein coupled receptors and cytokine receptors, also induce association between EGFR and c-Src and subsequent oncogenic consequences of this interaction. Because of their important roles in the etiology and progression of a broad spectrum of cancers, EGFR and c-Src represent signaling molecules that are ripe for combinatorial therapeutic targeting.