P190RhoGAP prevents mitotic spindle fragmentation and is required to activate Aurora A kinase at acentriolar poles.

Assembly of the mitotic spindle is essential for proper chromosome segregation during mitosis. Maintenance of spindle poles requires precise regulation of kinesin- and dynein-generated forces, and improper regulation of these forces disrupts pole integrity leading to pole fragmentation. The formation and function of the mitotic spindle are regulated by many proteins, including Aurora A kinase and the motor proteins Kif2a and Eg5. Here, we characterize a surprising role for the RhoA GTPase-activating protein, p190RhoGAP, in regulating the mitotic spindle. We show that cells depleted of p190RhoGAP arrest for long periods in mitosis during which cells go through multiple transitions between having bipolar and multipolar spindles. Most of the p190RhoGAP-depleted cells finally achieve a stable bipolar attachment and proceed through anaphase. The multipolar spindle phenotype can be rescued by low doses of an Eg5 inhibitor. Moreover, we show that p190RhoGAP-depleted multipolar cells localize Aurora A to all the poles, but the kinase is only activated at the two centriolar poles. Overall, our data identify an unappreciated connection between p190RhoGAP and the proteins that control spindle poles including Aurora A kinase and Eg5 that is required to prevent or correct spindle pole fragmentation.

Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy.

Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired ß1 and ß3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.

A complex of p190RhoGAP-A and anillin modulates RhoA-GTP and the cytokinetic furrow in human cells.

The cytokinetic furrow is organized by the RhoA GTPase, which recruits actin and myosin II to the furrow and drives contractility. Here, we show that the RhoA GTPase-activting protein (GAP) p190RhoGAP-A (also known as ARHGAP35) has a role in cytokinesis and is involved in regulating levels of RhoA-GTP and contractility. Cells depleted of p190RhoGAP-A accumulate high levels of RhoA-GTP and markers of high RhoA activity in the furrow, resulting in failure of the cytokinetic furrow to progress to abscission. The loss of p190RhoGAP-A can be rescued by a low dose of the myosin II inhibitor blebbistatin, suggesting that cells fail cytokinesis because they have too much myosin activity. p190RhoGAP-A binds the cytokinetic organizer anillin, and mutants of p190RhoGAP-A that are unable to bind anillin or unable to inactivate RhoA fail to rescue cytokinesis defects in p190RhoGAP-A-depleted cells. Taken together, these data demonstrate that a complex of p190RhoGAP-A and anillin modulates RhoA-GTP levels in the cytokinetic furrow to ensure progression of cytokinesis.

The human papillomavirus E7 proteins associate with p190RhoGAP and alter its function.

UNLABELLED: 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.

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

INTRODUCTION: Src 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. METHODS: To 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. RESULTS: Src 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. CONCLUSIONS: Complete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.

Neuroendocrine-derived peptides promote prostate cancer cell survival through activation of IGF-1R signaling.

BACKGROUND: Neuroendocrine (NE) cells promote the progression of prostate cancer to a castration-resistant state through the production of paracrine growth factors. We have demonstrated this principle using in vitro and in vivo proliferative endpoints; however, the contributions of NE-derived pro-survival factors and anti-apoptosis to this phenomenon have not been thoroughly investigated. METHODS: Here, we utilized conditioned-medium (CM) from LNCaP cells, engineered to undergo NE differentiation, and examined its effects on PC3 and LNCaP cell survival. RESULTS: Statistically significant changes in clonogenic survival, Annexin V staining, PARP cleavage and trypan blue positivity of approximately twofold were observed in the presence of NE-derived CM relative to control-CM for both LNCaP and PC3 cells. These changes were partially abrogated by antagonists of the neuropeptides neurotensin, bombesin, and PTHrP. Selective inhibitors of IGF-1R, EGFR or Src caused significant and nearly complete blockade of prostate cancer cell survival due to NE secretions. Similar increases in cell survival were observed for LNCaP or PC3 cells treated with NE-derived medium in the presence of docetaxel. Increased phosphorylation of IGF-1R, following treatment with NE-derived medium, was accompanied by decreased protein tyrosine phosphatase, receptor type F (PTPRF) mRNA, and protein levels. Overexpression of PTPRF decreased cell survival, the amplitude and duration of IGF-1R phosphorylation, and enhanced PARP cleavage in the presence of NE-derived medium. CONCLUSIONS: These data support the hypothesis that NE-derived factors act upon prostate cancer cells to stimulate pro-survival signaling and describe a novel mechanism of cross-talk between NE-derived factors and IGF-1R, mediated in part by PTPRF.

A mechanistic study of the effect of doxorubicin/adriamycin on the estrogen response in a breast cancer model.

OBJECTIVE: Estrogen treatment limits the cytotoxic effects of chemotherapy in estrogen receptor-positive (ER+) breast cancer cell lines, suggesting that estrogen pathway signaling may confer chemotherapeutic resistance. This study investigates the molecular responses of ER+ breast cancer cell lines to the chemotherapeutic agent, doxorubicin, in the presence or absence of estrogen. METHODS: ER+ MCF-7 and T47-D cells were cultured in hormone-starved or estrogen-containing media with or without doxorubicin at concentrations mimicking the low concentrations seen in plasma and tumor microenvironments in humans following typical bolus administration. Protein levels, phosphorylations, and interactions of estrogen-signaling molecules were assessed following these treatments, as well the effects of ER signaling inhibitors on cell proliferation. RESULTS: Surprisingly, estrogen and doxorubicin co-treatment markedly induced pro-growth alterations compared to doxorubicin alone and modestly enhanced estrogen alone-induced changes. Several inhibitors suppressed cell proliferation in the presence of doxorubicin and estrogen. CONCLUSIONS: These findings demonstrate that molecular changes caused by doxorubicin in ER+ breast cancer cells can be reversed by estrogen, providing molecular evidence for the poorer responses of ER+ tumors to doxorubicin in the presence of physiologic estrogen levels. Our results also suggest that the addition of drugs targeting the ER, EGFR, the SFKs, MEK, PI3K, and/or the MMP proteins to a conventional chemotherapy regimen may improve chemosensitivity.

c-Src and cooperating partners in human cancer.

The proto-oncogene c-src is rarely mutated in human cancers, and when overexpressed in normal cells is non- or weakly oncogenic. These observations have raised doubts about the involvement of c-src in the etiology of human tumors. However, recent studies have shown that c-Src, a non-receptor tyrosine kinase, exhibits elevated protein levels and activity in numerous types of human cancers. Furthermore, it has been found to be a critical component of multiple signaling pathways that regulate proliferation, survival, metastasis, and angiogenesis. Because of its important role in these oncogenic processes, it represents a therapeutic target ripe for exploitation.

Mitotic down-regulation of p190RhoGAP is required for the successful completion of cytokinesis.

p190RhoGAP-A (p190) is a GTPase-activating protein known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of Rho intrinsic GTPase activity. We have previously shown that p190 protein levels are cell cycle-regulated, decreasing in mitosis, and that this decrease is mediated by the ubiquitin-proteasome pathway. In addition, overexpression of p190 results in decreased RhoGTP levels at the cleavage furrow during cytokinesis, p190 and the RhoGEF Ect2 play opposing roles in cytokinesis, and sustained levels of p190 in mitosis are associated with cytokinesis failure, all findings that suggest but do not directly demonstrate that completion of cytokinesis is dependent on reduced levels of p190. Here we report, using an RNAi reconstitution approach with a degradation-resistant mutant, that decreased p190 levels are required for successful cytokinesis. We also show that the multinucleation phenotype is dependent on p190 RhoGAP activity, determine that the N-terminal GBDS1 region is necessary and sufficient for p190 mitotic ubiquitination and degradation, and identify four N-terminal residues as necessary for the degradation of p190 in mitosis. Our data indicate that in addition to activation of RhoGEF(s), reduction of RhoGAP (p190) is a critical mechanism by which increased RhoGTP levels are achieved in late mitosis, thereby ensuring proper cell division.

Epidermal growth factor receptor translocation to the mitochondria: regulation and effect.

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

In vitro and in vivo histone deacetylase inhibitor therapy with vorinostat and paclitaxel in ovarian cancer models: does timing matter?

OBJECTIVE: To determine whether the combination of vorinostat (suberoylanilide hydroxamic acid, SAHA) and paclitaxel is more effective than either individual agent and to evaluate the effect of drug sequencing in ovarian cancer cell lines and in mouse models. METHODS: For in vitro studies, three ovarian cancer cell lines (2774, SKOV-3 and OVCAR-3) were grown with either 10 nM paclitaxel, or vorinostat (0.3, 1, 3 or 10 µM), or vehicle (DMSO) and subsequently treated with 10 nM paclitaxel, or vorinostat (0.3, 1, 3, or 10 µM), or DMSO. Apoptosis was analyzed. In the mouse, treatments were given for a total of 5 weeks: vehicle control, paclitaxel, vorinostat, vorinostat followed by paclitaxel, paclitaxel followed by vorinostat, and simultaneous vorinostat and paclitaxel. Endpoints were survival, ascites and tumor weight. Protein expression was analyzed by Western blots. RESULTS: In two cell lines (SKOV-3, OVCAR-3), the sequence of vorinostat and paclitaxel administration did not significantly alter the apoptosis percentages and the combination was not superior to individual agents. However, in one cell line (2774), the most effective combination in achieving apoptosis was paclitaxel followed by low dose vorinostat. In the mouse model, both control and vorinostat alone treatment groups were inferior to paclitaxel and the combination treatment groups, but no significant differences were observed between the groups receiving both paclitaxel and vorinostat based on the sequence of administration. CONCLUSIONS: The efficacy of the combination and sequence of vorinostat and paclitaxel administration was cell line dependent and suggests that responses vary based on tumor specific characteristics.

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.

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.

Androgen-independent growth and tumorigenesis of prostate cancer cells are enhanced by the presence of PKA-differentiated neuroendocrine cells.

The neuroendocrine status of prostatic adenocarcinomas is considered a prognostic indicator for development of aggressive, androgen-independent disease. Neuroendocrine-like cells are thought to function by providing growth and survival signals to surrounding tumor cells, particularly following androgen ablation therapy. To test this hypothesis directly, LNCaP cells were engineered to inducibly express a constitutively activated form of the cyclic AMP-dependent protein kinase A catalytic subunit (caPKA), which was previously found upon transient transfection to be sufficient for acquisition of neuroendocrine-like characteristics and loss of mitotic activity. Clonal cells that inducibly expressed caPKA enhanced the growth of prostate tumor cells in anchorage-dependent and anchorage-independent in vitro assays as well as the growth of prostate tumor xenografts in vivo, with the greatest effects seen under conditions of androgen deprivation. These results suggest that neuroendocrine-like cells of prostatic tumors have the potential to enhance androgen-independent tumor growth in a paracrine manner, thereby contributing to progression of the disease.

A window-of-opportunity clinical trial of dasatinib in women with newly diagnosed endometrial cancer.

OBJECTIVE: To determine the extent of dasatinib uptake and effect on Src kinase activity in tumor, normal adjacent tissue, and blood in newly diagnosed endometrial cancer patients. METHODS: Dasatinib was dosed at 100 or 200 mg PO BID at 32 and 8 h preoperatively. Blood and tissue were collected pre-treatment and at surgery to assess active (pY419) and total Src protein (pharmacodynamics [PD]) and pharmacokinetics (PK). Plasma PK and PD were also analyzed at 2, 4 and 8 h following the second dose. RESULTS: Ten patients completed the study, 5 at each dose level (DL). Average (median, standard deviation, range) 2 h plasma concentration of drug was 119 (121, 80, 226) and 236 (162, 248, 633) ng/mL, for the 100 and 200 mg DL, respectively. Average ratio of 8 h normal and tumor tissue to plasma concentration overall was 3.6 (2.3, 3.4, 9.6) and 8.3 (3.2, 11.9, 38.7), respectively. Dasatinib concentration in tumor was higher than in plasma for both DL. Four patients displayed significant reductions in pTyr419Src at ≥ 1 time points in blood, and four patients satisfied the PD activity criteria in tissue, with reductions in pTyr419Src of ≥ 60%. CONCLUSIONS: This is the first study to show PK and PD effects of dasatinib in tumor tissue, allowing evaluation of tissue PD markers as a function of tumor dasatinib concentration. Dasatinib tissue concentrations at 8 h after dosing were associated with modulation of pTyr419Src, total Src protein, and pTyr419Src/Src ratio. All patients had reduction in at least one Src parameter in either tissue or blood.

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.

Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving epidermal growth factor receptor and signal transducer and activator of transcription 5b.

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II.

When co-overexpressed, the epidermal growth factor receptor (EGFR) and c-Src cooperate to cause synergistic increases in EGF-induced DNA synthesis, soft agar colony growth, and tumor formation in nude mice. This synergy is dependent upon c-Src-mediated phosphorylation of a unique tyrosine on the EGFR, namely, tyrosine 845 (Y845). Phenylalanine substitution of Y845 (Y845F) was found to inhibit EGF-induced DNA synthesis without affecting the catalytic activity of the receptor or its ability to phosphorylate Shc or activate mitogen-activated protein kinase. These results suggest that synergism may occur through alternate signaling pathways mediated by phosphorylated Y845 (pY845). One such pathway involves the transcription factor Stat5b. Here we describe another pathway that involves cytochrome c oxidase subunit II (CoxII). CoxII was identified as a specific binding partner of a pY845-containing peptide in a phage display screen. EGF-dependent binding of CoxII to the wild type but not to the mutant Y845F-EGFR was confirmed by coimmunoprecipitation experiments. This association also required the kinase activity of c-Src. Confocal microscopy, as well as biochemical fractionation, indicated that the EGFR translocates to the mitochondria after EGF stimulation, where it colocalizes with CoxII. Such translocation required the catalytic activity of the receptor but not phosphorylation of Y845. However, ectopic expression of the Y845F-EGFR prevented the EGF from protecting MDA-MB-231 breast cancer cells from adriamycin-induced apoptosis, whereas two mutants of Stat5b, a dominant-interfering mutant (DNstat5b) and a tyrosine mutation at 699 (Y699F-Stat5b) did not. Taken together, these data suggest that, through the ability of EGFR to translocate to the mitochondria, the binding of proteins such as CoxII to pY845 on the EGFR may positively regulate survival pathways that contribute to oncogenesis.

Estrogen negatively regulates epidermal growth factor (EGF)-mediated signal transducer and activator of transcription 5 signaling in human EGF family receptor-overexpressing breast cancer cells.

Breast cancer cell growth may be stimulated by 17beta-estradiol (E2) or growth factors like epidermal growth factor (EGF). However, tumors typically depend on only one of these pathways and may overexpress either estrogen receptor (ER) or EGF receptor (EGFR) and related family members. Tumors overexpressing EGFR are more aggressive than those expressing ER. Intracellular mediators of these growth-stimulatory pathways are not completely defined, but one potential common mediator of EGF and E2 signaling is the transcription factor signal transducer and activator of transcription 5 (STAT5). To investigate the role of STAT5 in potential crosstalk between E2 and EGF, MDA-MB231 and SKBr3 breast cancer cells, which are ER-negative and overexpress human EGF family receptors, were used. Introduction of ERalpha and treatment with E2 decreased EGF-induced tyrosine phosphorylation of STAT5b, basal and EGF-induced STAT5-mediated transcription, and EGF-stimulated DNA synthesis in these cells. Suppressive effects of E2-EpsilonRalpha were specific for STAT5, as EGF stimulation of MAPK was unaffected. Deletion/mutation analysis of ERalpha demonstrated that the DNA-binding domain was insufficient, and that the ligand-binding domain was required for these responses. ERalpha transcriptional activity was not necessary for suppression of STAT5 activity. Overexpression of c-Src did not prevent suppression of STAT5 activity by E2 and ERalpha. However, ERalpha did prevent basal increases in STAT5 activity with overexpressed c-Src. In the context of human EGF receptor family overexpression, E2-ER opposes EGF signaling by regulating STAT5 activity. STAT5 may be a crucial point of signaling for both E2 and growth factors in breast cancer cells, allowing targeted therapy for many types of breast tumors.

Src family kinases, key regulators of signal transduction.

The Src family of protein tyrosine kinases (SFKs) plays key roles in regulating signal transduction by a diverse set of cell surface receptors in the context of a variety of cellular environments. SFKs have evolved many ingenious molecular strategies to couple receptors with the cytoplasmic signaling machinery. The contributions to this issue of ONCOGENE describe how this machinery regulates fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals. The pleiotropic functions of Src and Src family members underscore the importance of these kinases and explain why many of the members of this family have been identified as cellular oncogenes. In this volume, we have attempted to provide the reader with an overview of the current understanding of the function of Src family kinases in the regulation of selected cellular signaling pathways.