Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts.

Focal adhesion kinase (FAK) is a key mediator of integrin signaling, which has been implicated in the regulation of cell migration and cell cycle progression. Using chimeric molecules that fuse the focal adhesion targeting (FAT) sequence directly to several signaling molecules, we investigated the potential role of FAK recruitments of signaling molecules to focal contacts in the regulation of cell migration and cell cycle progression. We found that fusion of FAT to Src, the p85 subunit of phosphatidylinositol 3-kinase, Grb7 and Grb2 resulted in the efficient focal adhesion targeting of these signaling molecules. We showed that expression of Src-FAT, p85-FAT, or Grb7-FAT, but not Grb2-FAT, each stimulated cell migration. Interestingly, tyrosine phosphorylation of paxillin, but not p130cas, was induced by expression of Src-FAT, suggesting a potential role of paxillin in mediating stimulation of cell migration by the chimeric molecule. In contrast, targeting of Grb2, but not Src, p85, or Grb7, to focal contacts increased cell cycle progression. Biochemical analyses correlated Erk activation by Grb2-FAT with its stimulation of cell cycle progression. Together, these results suggest that at least part of the role of FAK interaction with these signaling molecules is to recruit them to focal contacts and that distinct FAK signaling complexes are involved in the regulation of cell migration vs. cell cycle progression.     

Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 human breast cancer cell line

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. The mechanism underlying the increased proliferation could involve the induction of components of the insulin-like growth factor signal transduction pathway by estrogen. In this study we have examined the regulation of the expression of insulin receptor substrate-1, a major intracellular substrate of the type I insulin-like growth factor receptor tyrosine kinase. Estradiol increased insulin receptor substrate-1 mRNA and protein levels at concentrations consistent with a mechanism involving the estrogen receptor. Insulin receptor substrate-1 was not induced significantly by the antiestrogens tamoxifen and ICI 182,780, but they inhibited the induction of insulin receptor substrate-1 by estradiol. Analysis of tyrosine-phosphorylated insulin receptor substrate-1 showed that the highest levels were found in cells stimulated by estradiol and insulin-like growth factor-I, whereas low levels were found in the absence of estradiol irrespective of whether type I insulin-like growth factor ligands were present. Insulin receptor substrate-2, -3, and -4 were not induced by estradiol. These results suggest that estrogens and antiestrogens may regulate cell proliferation by controlling insulin receptor substrate-1 expression, thereby amplifying or attenuating signaling through the insulin-like growth factor signal transduction pathway.     

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.     

Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain

Grb14 is an adapter protein that is known to be overexpressed in estrogen receptor positive breast cancers, and in a number of prostate cancer cell lines. Grb14 has been demonstrated to bind to a number of activated receptor tyrosine kinases (RTKs) and to modulate signals transduced through these receptors. The RTKs to which Grb14 binds include the insulin receptor (IR), the fibroblast growth factor receptor (FGFR), the platelet-derived growth factor receptor (PDGFR), and the tunica endothelial kinase (Tek/Tie2) receptor. Grb14 has been shown to bind to these activated RTKs through its Src homology 2 (SH2) domain, with the exception of the insulin receptor, where the primary binding interaction is via a small domain adjacent to the SH2 domain (the BPS or PIR domain). Grb14 is a member of the Grb7 family of proteins, which also includes Grb7 and Grb10. We have solved the solution structure of the human Grb14-SH2 domain and compared it with the recently determined Grb7-SH2 and Grb10-SH2 domain structures.     

The adapter protein ZIP binds Grb14 and regulates its inhibitory action on insulin signaling by recruiting protein kinase Czeta

Grb14 is a member of the Grb7 family of adapters and acts as a negative regulator of insulin-mediated signaling. Here we found that the protein kinase Czeta (PKCzeta) interacting protein, ZIP, interacted with Grb14. Coimmunoprecipitation experiments demonstrated that ZIP bound to both Grb14 and PKCzeta, thereby acting as a link in the assembly of a PKCzeta-ZIP-Grb14 heterotrimeric complex. Mapping studies indicated that ZIP interacted through its ZZ zinc finger domain with the phosphorylated insulin receptor interacting region (PIR) of Grb14. PKCzeta phosphorylated Grb14 under in vitro conditions and in CHO-IR cells as demonstrated by in vivo labeling experiments. Furthermore, Grb14 phosphorylation was increased under insulin stimulation, suggesting that the PKCzeta-ZIP-Grb14 complex is involved in insulin signaling. The PIR of Grb14, which also interacts with the catalytic domain of the insulin receptor (IR) and inhibits its activity, was preferentially phosphorylated by PKCzeta. Interestingly, the phosphorylation of Grb14 by PKCzeta increased its inhibitory effect on IR tyrosine kinase activity in vitro. The role of ZIP and Grb14 in insulin signaling was further investigated in vivo in Xenopus laevis oocytes. In this model, ZIP potentiated the inhibitory action of Grb14 on insulin-induced oocyte maturation. Importantly, this effect required the recruitment of PKCzeta and the phosphorylation of Grb14, providing in vivo evidences for a regulation of Grb14-inhibitory action by ZIP and PKCzeta. Together, these results suggest that Grb14, ZIP, and PKCzeta participate in a new feedback pathway of insulin signaling.     

Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression

Focal adhesion kinase (FAK) is an important mediator of signal transduction pathways initiated by integrins in cell migration, survival and cell cycle regulation. The ability of FAK to mediate integrin signaling in the regulation of cell cycle progression depends on the phosphorylation of Tyr397, which implies a functional significance for the formation of FAK signaling complexes with Src, phosphatidylinositol-3-kinase (PI3K) and Grb7. We have previously described a FAK mutant, D395A, that selectively disrupts FAK binding to PI3K, but allows FAK association with Src. Using this mutation in a mislocalized FAK mutant background, we show here that formation of a FAK/PI3K complex is not sufficient for cell cycle progression but the formation of a FAK/Src complex plays an essential role. We also show that mutation of D395 to A disrupted FAK association with Grb7. This suggests that a FAK/Grb7 complex is not involved in the cell cycle regulation either, which is supported by direct analysis of cells expressing a dominant negative Grb7 construct. Finally, we provide evidence that the Src-dependent association of FAK with Grb2 and p130(Cas) are both required for the regulation of cell cycle progression by FAK. Together, these studies identify important FAK downstream signaling pathways in cell cycle regulation.     

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression.

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.     

Grb10 mediates insulin-stimulated degradation of the insulin receptor: a mechanism of negative regulation

Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.     

BMP-6 inhibits cell proliferation by targeting microRNA-192 in breast cancer

Although bone morphogenetic protein-6 (BMP-6) has been identified as a tumor suppressor associated with breast cancer differentiation and metastasis, the potential roles of BMP-6 in regulating cell cycle progression have not been fully examined. In the present study, we provide the novel finding that induction of BMP-6 in MDA-MB-231 breast cancer cells significantly inhibits cell proliferation by decreasing the number of cells in S phase of the cell cycle, resulting in inhibition of tumorigenesis in a nude mouse xenograft model. Further investigation indicated that BMP-6 up-regulates the expression of microRNA-192 (miR-192) in MDA-MB-231 cells. Elevated expression of miR-192 caused cell growth arrest, which is similar to the effect of BMP-6 induction. Importantly, depletion of endogenous miR-192 by miRNA inhibition significantly attenuated BMP-6-mediated repression of cell cycle progression. In breast cancer tissue, miR-192 expression is significantly down-regulated in tumor samples and positively correlates with the expression of BMP-6, demonstrating the inhibitory effect of BMP-6 on cell proliferation through miR-192 regulation. Additionally, using the RT² Profiler PCR Array, retinoblastoma 1 (RB1) was identified as a direct target of the BMP-6/miR-192 pathway in regulating cell proliferation in breast cancer. In conclusion, we have identified an important role for BMP-6/miR-192 signaling in the regulation of cell cycle progression in breast cancer. Furthermore, BMP-6/miR-192 was expressed at low levels in breast cancer specimens, indicating that this pathway might represent a promising therapeutic target for breast cancer treatment.     

Lipid-protein interactions of growth factor receptor-bound protein 14 in insulin receptor signaling

Many retinal degenerative diseases show an early loss of rod cells followed by cone cells. In these degenerations the pathological phenotype is apoptosis. We have previously demonstrated the light-dependent tyrosine phosphorylation of the insulin receptor in the retina, which leads to the activation of anti-apoptotic signaling molecules. The mechanism of the regulation of the insulin receptor in the retina is not known. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of the retinal insulin receptor (IRbeta) identified a member of the Grb7 (growth factor receptor-bound protein 7) gene family, Grb14. In this report, we describe the unique features of Grb14. Grb14 forms a specific complex with the cytoplasmic domain of IRbeta when both are expressed as hybrid proteins in yeast cells. This interaction is strictly dependent upon receptor tyrosine kinase activity. Deletion mutagenesis on Grb14 indicated a phosphorylated insulin receptor interacting (PIR) domain between the PH (pleckstrin homology) and SH2 (Src homology) domains that binds to IRbeta. Nuclear import assays in yeast indicated the presence of a functional nuclear localization signal in Grb14 between amino acids 63 and 68 (RRKKD). Subcellular localization of isolated retinas probed with anti-Grb14 antibody further confirmed the presence of Grb14 in nuclear fractions. Analysis using a protein-lipid overlay assay indicated binding of Grb14 and its PH domain to D3 phosphoinositides. In addition, Grb14-phosphoinositide 3,4,5-trisphosphate complexes are detected in lysates prepared from insulin-stimulated retina tissues, whereas Grb14-phosphoinositide 4,5-bisphosphate interactions are observed under non-insulin stimulated conditions. These findings suggest that Grb14 could be a diverse regulator of insulin receptor mediated pathways in the retina.     

BC-N102 suppress breast cancer tumorigenesis by interfering with cell cycle regulatory proteins and hormonal signaling,and induction of time-course arrest of cell cycle at G1/G0 phase

Mechanisms of breast cancer progression and invasion, often involve alteration of hormonal signaling, and upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Herein, we describe a rationally designed first-in-class novel small molecule inhibitor for targeting oncogenic and hormonal signaling in ER-positive breast cancer. BC-N102 treatment exhibits dose-dependent cytotoxic effects against ER+ breast cancer cell lines. BC-N102 exhibited time course- and dose-dependent cell cycle arrest via downregulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-Akt, CDK2, and CDK4 while increasing p38 mitogen-activated protein kinase (MAPK), and mineralocorticoid receptor (MR) signaling in breast cancer cell line. In addition, we found that BC-N102 suppressed breast cancer tumorigenesis in vivo and prolonged the survival of animals. Our results suggest that the proper application of BC-N102 may be a beneficial chemotherapeutic strategy for ER+ breast cancer patients.     

Grb10 adapter protein as regulator of insulin-like growth factor receptor signaling

Grb10 is a member of a superfamily of adapter proteins that includes Grb10, 7, 14, and a protein of Caenorhabditis elegans called Mig10. Grb10 proteins are binding partners for several trans-membrane tyrosine-kinase receptors, including the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR). Many recent reports have suggested a very important role of Grb10 in regulating IGF-IR signaling. In this review, we will focus on the role of Grb10 in IGF-I-induced mitogenesis and we will discuss the recent findings that show the involvement of Grb10 in the regulation of ligand-induced ubiquitination, internalization, and stability of the IGF-IR.     

Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.     

Accelerated mammary tumor development in mutant polyomavirus middle T transgenic mice expressing elevated levels of either the Shc or Grb2 adapter protein

The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling.     

Grb7 and Hax1 may colocalize partially to mitochondria in EGF‐treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1

Growth factor receptor bound protein 7 (Grb7) is a signal‐transducing adaptor protein that mediates specific protein–protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein‐binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras‐GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti‐apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor‐treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time‐dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.     

Divergent Roles for IRS-1 and IRS-2 in Breast Cancer Metastasis

The insulin receptor substrate (IRS) proteins are cytoplasmic docking proteins that function as essential signaling intermediates downstream of activated cell surface receptors, many of which have been implicated in breast cancer. The IRS proteins do not contain intrinsic kinase activity but rather function by organizing signaling complexes to initiate intracellular signaling cascades. IRS-1 and IRS-2 are expressed in normal mammary epithelial cells and in breast carcinoma cells, where they have been implicated in mediating signals to promote tumor cell survival, growth and motility. Although IRS-1 and IRS-2 are homologous, recent studies have revealed distinct functions for these adaptor proteins in regulating breast cancer progression. Specifically, IRS-2 is a positive regulator of metastasis, whereas IRS-1 may be a suppressor of metastasis. The observation that IRS-1 is inactivated in metastatic mammary tumors raises the possibility that IRS activity, rather than expression, may be a novel predictive indicator of metastasis. Understanding how the IRS proteins function in tumor progression is essential for future efforts aimed at developing approaches to target IRS-1 and IRS-2 in a diagnostic or therapeutic manner for the benefit of breast cancer patients.     

Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.